Prague: AD/PD Convenes Record Number of Youngsters and Leaders

17 Mar 2009

Last week, the capital city of the Czech Republic still lay dormant in its windy late-winter chill, but that did not keep neurodegenerative disease researchers from all over the world to bring to life a bustling six days of talks, posters, and ample exchange between young and established scientists. Perched atop a hill rising from the river Vltava (The Moldau, to classical music lovers), sits Prague’s conference center. A rather drab building, it nevertheless affords sweeping views over a beautiful city that is marked by the largest ancient castle in the world and beckoned conference-goers for walks and dinners. In addition to absorbing new science, AD/PD attendees learned that Prague is steeped in history not only of the political and cultural kind, but also, to the surprise of many, of the very origins of dementia research itself.

The 9th International Conference AD/PD 2009 took place 11-15 March following one and a half days’ worth of satellite meetings by European Union-funded research programs. The conference began amid some concern expressed by many scientists that the field at large might not be able to sustain two large conferences per year. The International Conference on Alzheimer’s Disease and Related Disorders (ICAD), which had previously alternated with the biennial AD/PD, will from 2009 on be held annually, starting four months from now in nearby Vienna, Austria. But AD/PD 2009 then turned out to draw 2,336 participants, some 150 more than its 2007 predecessor in Salzburg, Austria.

AD is organized jointly by Abraham Fisher of the Israel Institute for Biological Research in Ness-Ziona, Israel Hanin of Loyola University in Maywood, Illinois, Roger Nitsch of the University of Zurich, Switzerland, and Manfred Windisch of the Austrian contract research organization JSW-Research Ltd. in Graz. This year’s local organizers were Irena Rektorova of Masaryk University in Brno, Czech Republic, and Jakub Hort of Prague’s Charles University.

A non-scientific poll conducted among attendees (Material and Methods: ask everyone in sight, statistical analysis—none) generated the impression that they enjoyed the conference scientifically and socially, with unpublished data presented in a wide range of topic areas. People had ample opportunity to peruse the more than 1,100 posters, because they were split into two blocks and each poster stayed on its board for two days in areas near where the food was, so conference-goers could easily drift through the poster areas during breaks. Attendance at the afternoon sessions was high in part because the conference was fully catered, though many people understandably snuck off on occasion to explore Prague.

A theater show preceded the opening reception, and the program included an evening concert in four of the city’s churches, whose many towers evoke Prague’s nickname “City of 1,000 Spires.” This was a fitting outing in a city known for its active church music scene, though some quipped that sitting on a wooden bench in an unheated space after a day of having already sat through talks from 8:30 a.m. to 7 p.m. had a slight tinge of penance to it, too. (The perfect opportunity to ask absolution for that cutting manuscript review you wrote last month…). As always, AD/PD offered a dinner party with a live band and dancing. It was oversubscribed, but luckily Prague is a city where scientists who got turned away from the party could simply saunter around downtown and snap up inexpensive tickets for the State Opera’s performance of Giuseppe Verdi’s AIDA, which is what some did for a memorable night.

Junior scientists in particular flocked to this meeting, taking advantage of measures the organizers had implemented in response to feedback solicited in the closing session of the Salzburg conference. In particular, AD/PD 2009 awarded various types of award to young investigators. Though small in monetary value, they invited fresh faces onto the stage for recognition in the main plenary hall, and the conference program then dedicated three slide sessions exclusively to young investigator talks. Here are the names and affiliations of young scientists who won awards: Tania Alves, Fabio Canneva, Emanuela Colla, Tracey Dickson, Andre Fischer, Anat Frydman-Marom, Ilse Gijselinck, Cathleen Hanse, Arne Herring, Natsuki Kobayashi, Thomas Kukar, Madepalli Lakshmana, Seiko Nishimoto, Tiago Outeiro, Dominic Paquet, Laura Parkinnen, Rosa Rademakers, Lawrence Rajendran, Nora Scheinin, Maite Solas, John Henry Stockley, Katerina Venderova, Yipeng Wang, Donna Wilcock, Xiongwi Zhu.

Breakfasts featuring senior clinicians invited young investigators to network. Less well known, perhaps, is one further tool to convene the next generation. That is, the registration fee gets waived for one student from the lab of each invited speaker. This offer was not fully exploited this year, Fisher said, and will be more prominently advertised next time, when the conference is set to take place in Barcelona, Spain, in 2011.

“We young investigators got lots of support here, especially to meet the big shots in the field. It was a great opportunity to get both new science and social contact. And it’s the only meeting of this size where I get fed lunch every day,” Ling Li, of the University of Alabama in Birmingham, said at the farewell session.

Praise from scientists duly discharged, there were some quibbles, too. The quality of the talks varied at times as is true at most large conferences, and some noted that the topics of some sessions could have been more focused. Last-minute schedule changes—i.e., Dennis Selkoe, John Hardy, David Holtzman, Peter Lansbury, Mike Hutton, Christine van Broeckhoven and Marc Tessier-Lavigne—were spottily communicated, creating an element of surprise. “Half the time I go to a plenary talk, it’s not there,” joked Karen Duff of Columbia University in New York. “But then there’s the bonus of the unexpected talk. I actually got a lot out of this meeting.” Everyone who has ever organized a conference will sympathize with the challenges of scheduling busy people, and this minor kink will be ironed out next time, Fisher said.

The conference broke no major clinical news, but it did feature ample new data in multiple science areas. The title combination of Alzheimer and Parkinson diseases worked particularly well this year, said Christian Czech of Roche in Basel, Switzerland. Research into the considerable overlap at the clinical, the pathological, and the genetic level of a range of conditions that where traditionally categorized as either a dementing or a movement disorder is coming to the fore, and it is increasingly breaking down barriers between these formerly separate bastions of neurology. Check back for research stories to come live over the next few weeks.—Gabrielle Strobel.

Fifteenth-century astronomical clock on Prague's landmark Old Town Hall, founded in 1338. Image credit: Carmela Abraham

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Prague: What Say You, Alois—Should It Be “Alzheimer-Fischer” Disease?

20 Mar 2009

The 9th International AD/PD conference, held in Prague 11-15 March, made a big splash in the otherwise sleepy backwater of Alzheimer disease history. In the opening session of the meeting, Pavel Kalvach of the Charles University of Prague jolted jetlagged attendees with the news that his very city had hosted a contemporary of Alois Alzheimer’s who had described his rival’s eponymous disease in more depth than did Alzheimer himself. That seminal investigator was Oskar Fischer, and his story resonates with historical pain. Fischer’s contributions were widely noted and debated when he published them in 1907, 1910, and 1912, and for some years afterward. But they later became neglected as Fischer’s career crumbled amid nationalist tension and the anti-Semitism of his time. His life ended tragically in 1942 in Terezin (Theresienstadt), a concentration camp set up in a garrison town near Prague. This camp is especially known for having incarcerated noted artists, writers, musicians, scientists, and other scholars, whose cultural achievements in the camp the Nazis successfully touted as part of their propaganda campaign to hide the true horror of the camps, deceiving even the Red Cross on an invited visit in 1944.

Fischer remained consigned to oblivion, both in his home country and by most in the worldwide dementia community, until the fall of 2008, when Michel Goedert of the MRC laboratory of Molecular Biology in Cambridge, U.K., recounted in the journal Brain the story of what his visit to the Archives of Charles University, as well as conversations with Fischer’s descendants and present-day Czech researchers, brought to light. Freely available for download, Goedert’s paper makes for gripping reading about the historical context of Fischer’s life and also about how his observations intersect with those of other investigators at the time (Goedert, 2008).

“We are grateful to Goedert for this discovery. This country had completely forgotten Oskar Fischer,” Kalvach told the audience.

“News” to many, however, is rarely news to all. In Prague, conversation with other scientists turned up that Piet Eikelenboom, for one, wrote three years ago that Fischer’s three papers contain prescient conceptual insight into the role of inflammation in AD that found confirmation with the advent of new molecular techniques starting some 70 years later (Eikelenboom et al., 2006). A few other AD scientists have mentioned Fischer, as well, and even showed some of his drawings at talks at AD/PD (e.g., Gouras et al., 2005). At the conference, Kalvach brought Fischer’s story to a wide audience as part of his broader account on the early history of dementia research in Prague. Below are selected highlights.

Before Fischer
The early roots of psychiatry in the region of Bohemia reach back to the fifteenth century, Kalvach said. More detailed records exist of a “Tollhaus” (insane asylum) around the turn of the eighteenth century. Josef Riedel was the pre-eminent psychiatrist at the time, who established this field as an independent branch of academic medicine. His writings show that most patients received a diagnosis of “unknown,” and few lived long enough to provide much fodder for observation of age-related dementia. The region’s first magnum opus of psychiatric literature was a textbook by Karel Kuffner (1858-1940), written first in Czech and then translated to German. Kuffner focused on dementias, and debated Emil Kraepelin’s contemporaneous attempt to unify the different “Verbloedungsprocesse” (dementing processes) under a common designation, said Kalvach. All this predated the advent of neurology.

Yet another major figure in psychiatry also lived in Prague. Arnold Pick (1851-1924) is known today for his description of frontotemporal dementia and Pick’s disease. Pick published in English journals and attracted international specialists to Prague. (Curiously, Alzheimer discovered what was later called the Pick body, whereas Fischer, who worked under Pick for 16 years and pursued prolific interests, described no cases of FTD at all.) Pick headed Prague’s “Irrenanstalt” and the department of psychiatry at Prague’s German University (the city also had a Czech University), where Fischer was first assistant professor, then associate professor.

Fischer’s Time
Oskar Fischer was born into a German-speaking family in the town of Slany in Central Bohemia, some 16 miles northwest of Prague, in 1876. At age 24, he obtained a medical degree from the University of Prague, where he made seminal observations on dementia in his early thirties. The university had been divided into separate German and Czech institutions in the course of Czech nationalism against the Austro-Hungarian Empire, which ruled what today is the Czech Republic until the end of World War 1. Under Pick or his successors, Fischer never obtained tenure at this university. After 17 years of research and many more of teaching, the university revoked Fischer’s appointment in January 1939, two months before the German invasion. As in Vienna, medical institutions quietly sympathetic with national socialist rule in Germany were preparing for the anticipated takeover by removing Jewish faculty, and inconvenient minds in general. According to Goedert’s paper, the German occupants also confiscated a sanatorium for the mentally ill that Fischer had co-founded in 1908 and led since then. Fischer continued a private practice until the Gestapo arrested him in 1941.

In 1907, the same year that Alois Alzheimer published his famous paper about the pathology and clinical course of Auguste Deter’s presenile dementia (see ARF Centennial news story), Oskar Fischer reported neuritic plaques in 12 of 16 cases of senile dementia in the journal Monatsschrift fuer Psychiatrie and Neurologie, published by Karger. Alzheimer’s short paper was essentially a transcript of a lecture he had given the previous November in Tuebingen, Germany. Its undisputed achievement was the simultaneous description of both plaques and tangles in a clinically well-documented case of what would now be called early onset AD. Fischer’s first paper complements Alzheimer’s contribution by describing in great detail neuritic plaques and how they distort and push away nearby nerve fibers. Beyond that, it offers a detailed comparison of the 16 brains with dementia to 10 controls, 10 cases with psychosis, and 45 more with neurosyphilis, a predominant problem of neurology in these pre-antibiotic days.

Emil Redlich, a contemporary of Fischer’s in Vienna, had first used the term “plaque” in his publications, but he thought they resulted from proliferating glia. Fischer in his 1907 paper wrote, “Many circumstances contest this view.” Rather, he viewed the plaques as necrotic inclusions associated with abnormal neurites containing “neurofibrils.” Clinically, he called those 12 cases “presbyophrenia,” a type of dementia marked by confabulations, disorientation, memory impairment, hyperactivity, and elevated mood. This term remained in use until about 1930 but then faded when dementia was redefined. The perception of Fischer’s clinico-pathological work was tied to this term, explaining in part why his work could have been ignored later on.

In his 1910 paper, Fischer reported his study of 275 brains of people with various conditions, many of them older than 50 years. Fifty-six cases from among this old cohort had plaques, some had tangles, and the majority fit his clinical criteria of presbyophrenia. In an uncanny expression of a debate that continues to this day, he noticed that some cases showed plaque pathology but no or only mild clinical symptoms. Fischer speculated that, had these people lived longer, they would have developed the full clinical disease and more abundant pathology. But “he was aware of the fact that the frequent occurrence of abundant plaques in old people without mental impairment could fatally undermine his view that the plaque represents the morphological substrate of presbyophrenic dementia,” Goedert writes. By 1912, Fischer reported having found plaques in two cases of 35 normal old people, and he considered these people to have had presymptomatic disease. This issue is undergoing a direct test only today, as longitudinal studies track live amyloid imaging in cognitively normal elderly people.

But even in these early years, Fischer and his contemporaries already debated whether plaques occur with normal aging or are pathological. “Fischer was remarkably modern,” writes Goedert. “He separated dementia from normal aging and considered clinical signs not to be decisive by themselves for diagnosis.” Instead, he saw plaque pathology as the defining criterion.

“Fischer’s originality was enormous,” Kalvach said in Prague. Fischer was the first to describe cerebral amyloid angiopathy, or CAA. He classified eight stages of plaque development, of which stage six described plaque deposits around the walls of blood vessels. He did not foresee a connection between vascular amyloid and hemorrhages, though, Goedert noted.

Fischer placed plaque formation in the larger context of inflammation, foreshadowing a concept that would later be validated but which he lacked tools to pursue experimentally. In 1910, Fischer wrote that plaques form as the result of an extracellular deposition of an abnormal substance in the cortex that induces a local inflammatory reaction, followed by an attempted but doomed regenerative response of the surrounding nerve fibers. In their 2006 review on neuroinflammation in AD, Eikelenboom and colleagues at University of Amsterdam and Vrije University Medical Center, also in that city, recount how Fischer unsuccessfully searched plaques for cellular evidence of inflammation and for complement activation. Microglia were barely understood at the time, and Fischer’s idea remained obscure until the 1980s, when the advent of monoclonal antibodies for immunohistochemistry revived interest in neuroinflammation.

At that time, Eikelenboom detected the early complement proteins C1q, C3, and C4 in senile plaques (Eikelenboom and Stam, 1982) and later showed that they reflected complement activation. Other labs, including Pat McGeer’s, Joe Roger’s, and Annemieke Rozemuller’s, continued this line of research, reporting full-bore complement activation and clusters of activated microglia at plaques. Today, the notion of chronic inflammatory processes surrounding amyloid pathology has become widely accepted. Last but not least, Fischer formulated another concept that present-day scientists validated. He spotted signs of an attempt by neurites to sprout and regenerate near plaque lesions, an idea developed by Carl Cotman and Thomas Arendt.

As for Alzheimer, in his 1911 paper he confirmed Fischer’s 1907 discovery of the neuritic plaque. Both scientists had some disagreements about how to interpret their findings, but they shared other interests, such as their work on neurosyphilis and their humane and scientific stance regarding the ravages the Great War was wreaking on the psyche of soldiers. But Alzheimer was unable to pursue debate with Fischer, or help establish his younger rival’s legacy, because he died in 1915 at age 51 (see ARF related news story).

During Fischer’s most productive time in Prague, one young Albert Einstein worked just a few blocks from him for a few years, Kalvach told the audience. Many other great thinkers in physics and medicine got out in time and established flourishing careers in their new countries, leaving behind an impoverished intellectual scene in central Europe, from which cities such as Vienna and Prague took decades to recover. Would that Fischer could have been so lucky.—Gabrielle Strobel

Fourteenth-century synagogue in Prague, with statue of Moses. Image credit: Benjamin Wolozin

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References

News Citations

Paper Citations

Goedert M. Oskar Fischer and the study of dementia. Brain. 2009 Apr;132(Pt 4):1102-11. PubMed.
Eikelenboom P, Veerhuis R, Scheper W, Rozemuller AJ, Van Gool WA, Hoozemans JJ. The significance of neuroinflammation in understanding Alzheimer's disease. J Neural Transm. 2006 Nov;113(11):1685-95. PubMed.
Gouras GK, Almeida CG, Takahashi RH. Intraneuronal Abeta accumulation and origin of plaques in Alzheimer's disease. Neurobiol Aging. 2005 Oct;26(9):1235-44. PubMed.
Eikelenboom P, Stam FC. Immunoglobulins and complement factors in senile plaques. An immunoperoxidase study. Acta Neuropathol. 1982;57(2-3):239-42. PubMed.

Prague: N-terminal APP Story Buzzes in the Hallways

24 Mar 2009

A story that broke at Keystone last month strongly pervaded the 9th International AD/PD conference in Prague, popping up in presentations and hallway chatter even before its lead investigator, Marc Tessier-Lavigne of Genentech in South San Francisco, California, spoke in the final session of the AD/PD conference. Tessier-Lavigne, whose summary and comments are at ARF Keystone story, reported no new data beyond those published on 19 February. But listening to scientists discuss it in Prague, it appears that the finding has stirred up the field of Alzheimer disease research at a time when many investigators are anxiously watching the progress of the current crop of anti-amyloid medicines in clinical trials. Hence, here is a brief summary of the thoughts expressed, questions to pursue.

In a nutshell, the Genentech scientists reported that an N-terminal cleavage product of APP binds the death receptor 6 (DR6) to trigger neurodegeneration in neurons starved of trophic support. Questions that bubbled up about this work include these: Is this pathway at play in AD? If so, does it diminish the importance of Aβ’s effect on synapses? Will both pathways be important? Are they connected? Does this science imply Aβ monotherapy trials won’t show efficacy?

The topic came up in talks, as well. To quote but two examples, Dennis Selkoe of Brigham and Women’s Hospital in Boston wrapped up a plenary lecture on the amyloid hypothesis with the remark that the Genentech work provides strong evidence for a normal physiological function of APP’s 35kD N-terminal fragment in axonal pruning during development, and that its relevance to AD remains to be shown. Martin Citron of Eli Lilly and Company in Indianapolis, Indiana, who cloned BACE with Bob Vassar and colleagues, noted in his talk that if this AD relevance indeed becomes clear, then that would make BACE an even more attractive drug target. That is because BACE inhibitors would reduce production of the APP N-terminal fragment as well as of Aβ. Such drugs could slow down both the proposed pathway of DR6-mediated neurodegeneration and the synaptic effects of Aβ, as both lie downstream from BACE cleavage. In contrast, γ-secretase inhibition would make no dent in the production of the troublesome N-terminal APP fragment. That said, Citron added, BACE continues to humble drug developers. After nine years of effort since the structure of BACE was solved, aided by the tailwind of knowledge gained during drug development against the similar enzyme HIV protease in the 1990s, only two BACE inhibitors have entered early-stage clinical trials to date, Citron noted (see ARF conference story).

More broadly, the Genentech paper has refocused the field’s attention on the nagging fact that it has been unable to establish a strong and direct connection between Aβ and neurodegeneration in vivo, even though neurodegeneration remains a defining feature of the human disease. The absence of frank neuronal death in APP- and PS1-transgenic mice has receded from view in recent years. Partly, researchers felt that the mice model early stages of AD, partly that they don’t live long enough for the neurons to die. Another possible explanation is that the models lack a still-unknown component of AD, for example, complement proteins that hasten neuronal death in humans but not mice, as suggested by Pat McGeer (for more on complement in AD and mouse models, see report of Bar Harbor Workshop). Even as these questions remained unresolved, research on synaptic biology and behavior started showing disruptive effects of various forms of Aβ oligomer in the hands of several independent labs. Indeed at AD/PD, Karen Ashe of the University of Minnesota in Minneapolis presented in her plenary lecture unpublished data on the toxicity of Aβ oligomers isolated from human AD brains. As this line of research has become increasingly prominent, it has fed a gradual redefinition of Alzheimer’s as a synaptic disease, with less emphasis on axonal and somatic degeneration.

Yet other voices have been cautioning for some time that Aβ explains only part of what goes awry in late-onset AD. John Hardy of University College, London, U.K., voiced a similar concern at a satellite symposium preceding AD/PD. Though Hardy is widely seen to have co-formulated the amyloid hypothesis, he has been reminding the field in recent years that the hypothesis still needs to plug the two stubborn knowledge gaps of a normal function of APP and the cause of neuronal death. “The amyloid hypothesis is not wrong but more complicated in ways that are difficult to deal with. The debate about what is the toxic species—plaque or oligomers—skirts the problem that none of them are toxic enough to cause neurodegeneration. That the mouse models have great plaques but almost no neurodegeneration should still concern us,” Hardy said.

The Genentech study prompted many questions for future study. Here is a sampling: Several scientists, including Frank LaFerla at University of California, Irvine, asked why APP-overexpressing mice do not show massive neurodegeneration if the 35kD fragment is so potent? After all, the precursor for the N-terminal fragment sits on the neural membrane in excess amount, and BACE is active. The answer might be found in the trophic factor dependence of the DR6 pathway, others said. Tessier-Lavigne emphasized that only starved neurons are vulnerable. The soluble N-terminal product of α-secretase shedding is neurotrophic, hence might extend a level of protection to these mice. Indeed, some of the earliest strains of hAPP-transgenic mice, made by Carmela Abraham of Boston University and Lennart Mucke of the Gladstone Institute in San Francisco drew little attention because their level of overexpression was insufficient to generate plaques, but these mice were notably protected in various models of neuronal injury (Mucke et al., 1994; Mucke et al., 1995; Masliah et al., 1997).

Another question that prompted curiosity was what kept the N-termini of APP to activate the DR6 receptor on neighboring axons running closely packed alongside each other. Other people asked what the role was, if any, of the APLPs, APP dimerization, or binding of APP to lipoprotein receptors, and whether the immediate N-terminal product of BACE cleavage, sAPPβ, could activate DR6, as well.

Abraham wondered what might be the identity of the mystery protease that cuts the 35 kD DR6 ligand out of sAPPβ. APP itself might provide a clue there, she said, as it comes in two flavors. The longer APP751 occurs in many cell types, and its N-terminal region contains the Kunitz protease inhibitor (KPI) domain, which is active against serine proteases (e.g., Hook et al., 1999). Neurons tend to express the shorter APP695 form without the KPI domain. If experiments show that only N-termini from the shorter APP activate DR6, that might point investigators toward which type of protease to look for, Abraham said. Incidentally, KPI’s role in blood clotting (Van Nostrand et al., 1992) was the first physiological function discovered for APP, and deserves renewed attention, Hardy said.

As for Tessier-Lavigne himself, he said after the lecture that if future experiments demonstrate a role of the DR6 pathway in AD, that role may well turn out to complement the amyloid hypothesis, not necessarily replace it. More than one pathway can operate in AD, and the notion, for example, that one leads from APP’s N-terminus to axonal degeneration while another leads from Aβ to synaptic impairment is not mutually exclusive. The new pathway at present has no evidence at all to explain why presenilin mutations cause AD, and presenilin’s functions and γ-secretase’s collective substrates will have to be examined to address this question.

All things considered, does Genentech’s foray into APP signaling mean clinical trials based on anti-amyloid strategies should not proceed? “Absolutely not,” Tessier-Lavigne said. “In fact, Genentech has an anti-Aβ antibody in Phase 1, and the trial continues apace.” (See Drugs in Clinical Trials.) “The antibodies that are further ahead have potential liabilities, in terms of either safety or efficacy, that might jeopardize this line of clinical investigation before the amyloid hypothesis has been truly put to the test,” Tessier-Lavigne added. Other clinicians voiced this concern at the AD/PD conference, as well.

What is clear, however, is that even at this early stage of research, the 35 kD N-terminal fragment of APP represents a potential new drug target worth exploring, Tessier-Lavigne added. It is a type of target that pharma and biotech companies have successfully exploited before, for example, with drugs such as infliximab, etanercept, and adalimumab. These biologics target tumor necrosis factor α, which, like the 35 kD fragment, is a ligand for the superfamily of receptors that includes DR6.—Gabrielle Strobel.

The "Transparency 2009" statue accompanies the Czech Republic's current EU presidency. For a night view, check Radio Praha. Image credit: Benjamin Wolozin

References

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Mucke L, Masliah E, Johnson WB, Ruppe MD, Alford M, Rockenstein EM, Forss-Petter S, Pietropaolo M, Mallory M, Abraham CR. Synaptotrophic effects of human amyloid beta protein precursors in the cortex of transgenic mice. Brain Res. 1994 Dec 15;666(2):151-67. PubMed.
Mucke L, Abraham CR, Ruppe MD, Rockenstein EM, Toggas SM, Mallory M, Alford M, Masliah E. Protection against HIV-1 gp120-induced brain damage by neuronal expression of human amyloid precursor protein. J Exp Med. 1995 Apr 1;181(4):1551-6. PubMed.
Masliah E, Westland CE, Rockenstein EM, Abraham CR, Mallory M, Veinberg I, Sheldon E, Mucke L. Amyloid precursor proteins protect neurons of transgenic mice against acute and chronic excitotoxic injuries in vivo. Neuroscience. 1997 May;78(1):135-46. PubMed.
Hook VY, Sei C, Yasothornsrikul S, Toneff T, Kang YH, Efthimiopoulos S, Robakis NK, Van Nostrand W. The kunitz protease inhibitor form of the amyloid precursor protein (KPI/APP) inhibits the proneuropeptide processing enzyme prohormone thiol protease (PTP). Colocalization of KPI/APP and PTP in secretory vesicles. J Biol Chem. 1999 Jan 29;274(5):3165-72. PubMed.
Van Nostrand WE, Schmaier AH, Wagner SL. Potential role of protease nexin-2/amyloid beta-protein precursor as a cerebral anticoagulant. Ann N Y Acad Sci. 1992 Dec 31;674:243-52. PubMed.

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Prague: Tau-Laden Neurons in Zebrafish Glow, Perish on Candid Camera

26 Mar 2009

Updated 15 April 2009. The protein tau has for years bedeviled dementia researchers. Critically important in Alzheimer’s and many frontotemporal dementias, it has nonetheless been a slippery drug target. The basics of its wayward ways have been clear for decades: hyperphosphorylation followed by neurofibrillary tangle pathology that spreads in lockstep with disease progression. Yet the crucial details of exactly what goes wrong early on, and exactly how to set it right, have remained stubbornly elusive in vivo. At the 9th International Conference AD/PD 2009, held 11-15 March in Prague, researchers from Germany and Sweden presented a new model that they hope will change that.

The model is a transgenic zebrafish that develops tauopathy and death of flaming red neurons over the course of six days, and that responds to new tau kinase inhibitors. It is the result of a four-year collaboration between Dominik Paquet and colleagues in the laboratories of Christian Haass at the Ludwig-Maximilians University in Munich, neurogeneticist Reinhard Koester at the Helmholtz Zentrum in that city, Eva-Maria Mandelkow at the Max-Planck Unit for Structural Molecular Biology in Hamburg, and Ratan Bhat of AstraZeneca Research & Development in Södertälje, Sweden.

Danio rerio is a small aquarium fish that was touted as the new wave in vertebrate molecular biology some years ago, when genetic manipulation of the minnow became tractable. Ease of use was the mantra: because they remain translucent far into their development, zebrafish can literally develop in plain view under the microscope; because embryos and larvae thrive in 96-well plates, quasi high-throughput drug screens are an option; because they live in water, the scientist can simply drip their favorite compound into it and see if the fish develop a phenotype—no fiddly injection into the tail vein, as in mice, for example. “Testing hypotheses in zebrafish is quick and easy,” co-author Bettina Schmid said during her presentation.

The trouble was that getting sufficiently high expression of transgenes proved anything but quick or easy, and as those robust models weren’t coming on line, zebrafish faded from view in neurodegeneration research even as they became an established model in developmental biology. This is where most of the development effort lay in this study, Haass said in an interview. The current model expresses the human tau-P301L mutation, which causes FDTP-17, in zebrafish neurons from a newly designed vector system. One of the vector’s tricks is that a driver construct activates transcription off of a second responder construct in two directions, such that both the transgene and a fluorescent reporter gene are expressed simultaneously. This means every embryo that lights up red under the microscope expresses human tau—no PCR needed. The other trick lies in adding a human transposase gene on either end of the constructs to increase the number of insertions. “We inject that into fertilized eggs, outcross the founders, and look for red embryos. That’s all. It’s simpler than making transgenic mice,” Schmid said.

The researchers constructed the vector with exchangeable cassettes, so tau can be replaced with other transgenes. In this vein, they have more recently made fish overexpressing TDP-43, α-synuclein, and Aβ, which took about three weeks per line. Making such a line with this existing vector requires no deep technical knowhow, Schmid noted; the bottleneck at present is that relatively few academic institutions, or pharma companies, maintain a zebrafish facility. The biotech company Zygogen in Atlanta, Georgia, holds some patents on tau-transgenic zebrafish, but these do not apply to academic research.

In Prague, Paquet showed that the tau fish start expressing early makers of tau phosphorylation, e.g., AT180, some 32 hours after fertilization. Using a panel of tau antibodies, he showed that later markers then followed, and binding for each marker built up over a couple of days. By seven days, late tau phosphorylation markers were fully expressed; by five weeks, the neurons contained flame-shaped, Gallyas-stained neurofibrillary tangles. “This sequence of tau changes reproduces, on a compressed time scale, what happens in humans,” Haass said.

Like some tau-transgenic mice (e.g., P301L; Lewis et al., 2000), these fish express tau in the spinal cord and consequently have a motor phenotype. Paquet showed a simple example, whereby normal fish dart away when pricked with a toothpick but tau-transgenics endure this indignity with nary a twitch of their little tails. Moreover, their spinal cord neurons grew out more slowly in vivo than is normal, probably because axonal transport deficits held up delivery of supplies to their growth cones.

But perhaps the most telegenic feature of this new model is that it affords a front-row seat to the sight of sick neurons dying in vivo. Paquet showed a time-lapse video sequence of a 10-hour live imaging session. He had added to the water the dye acridine orange, which enters only dying cells. Conference attendees watched how a red, i.e., P301L tau-bearing, neuron started balling up, blebbing its membrane, letting in the dye, and then disintegrating. “This is, to my knowledge, the first in-vivo live imaging of neuronal cell death in a vertebrate system,” Haass said. Besides filming the movie, the scientists also monitored cell death quantitatively, noting a massive increase relative to control fish by six days after fertilization.

Dying neuron lets in dye, turning yellow. (Movie link at end of story.) Image credit: Dominik Paquet

Finally, Paquet’s conference presentation included data about the possible application of this model for in-vivo validation of candidate drugs against the earliest step in the tauopathy cascade, i.e., tau hyperphosphorylation. Co-authors Bhat and colleagues at AstraZeneca have been using structure-based drug design of GSK3β co-crystallized with candidate inhibitors to come up with better inhibitors of that kinase, one of several known to phosphorylate tau (see also ARF Keystone story). GSK3β has been a drug target for many years, but previous inhibitors, such as AstraZeneca’s published compounds SB-216763 and SB-41526, were not safe enough for the clinic, and the widely prescribed human drug lithium chloride is neither very potent nor very selective. The new, unpublished compounds, AR164 and AR534, are potent in the desired low nanomolar range in vitro, and are also more selective than their predecessors for GSK3β. The new fish model came in handy for checking the compounds’ ability to cross the blood-brain barrier and show their moxy in a whole organism. (Yes, fish have a BBB, Paquet said; and yes, they also have GSK3β with a conserved active site.) Surprisingly, one of these new inhibitors performed as advertised, whereas the other did not, either because it bounced off the BBB or got degraded. This suggests that this zebrafish model can help to quickly eliminate the in-vivo duds from among a set of in-vitro candidates.

This might be a useful niche for drug studies, noted Haass. Independent industry commentators agreed that the zebrafish model is unsuitable for primary screens or to replace rodent testing, but that it could help select which in-vitro compounds to carry forward into more time-consuming and expensive rodent models. Moreover, the Munich group could test compounds that have entered clinical trials, such as AL-108/NAP or methylene blue. FDA-approved drugs could also be tested in this system for any unexpected effects on tauopathy. Finally, the system might enable scientists to address research questions, such as whether neurofibrillary tangles are necessary for neuronal death. This will be feasible once an ongoing technical upgrade from the current confocal setup to two-photon microscopy is complete, Haass said.

The movie elicited some oohs and aahs during the AD/PD session. When asked about the model, audience members mentioned some caveats, such as that the model is as artificial as overexpression models are in general, and that the tauopathy unfolds in the physiological context of a developing organism, not an aging one. The movie sequences can be viewed in the supplemental materials of the paper: Acridine uptake and death of P301L-expressing tau neuron; Motor phenotype after needle prod.—Gabrielle Strobel

View of Prague Castle, founded around 880, across the River Moldau. Image credit: Carmela Abraham

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Prague: Aβ Rehabilitated as an Antimicrobial Protein?

01 Apr 2009

If things go Robert Moir’s way, the weird and wonderful world of microbes may hand the field of Alzheimer disease research a physiological function for Aβ in the peptide’s 25th anniversary year (Glenner and Wong, 1984). In his talk at the 9th International Conference AD/PD, which unfolded 11-15 March in the Czech capital of Prague, Moir, a feisty Aussie at the Genetics and Aging Research Unit at Massachusetts General Hospital in Charlestown, came to the defense of the much maligned protein. Aβ is more than an evildoing piece of junk, Moir said, claiming that it defends the body day in and day out as part of the evolutionarily ancient innate immune system. “Aβ is a broad-spectrum antimicrobial peptide that shows bacteriostatic activity against clinically important organisms at concentrations similar to those of LL-37, an archetypal human AMP,” Moir said in his talk. Viewed from this angle, the properties Aβ has accrued in the AD literature would no longer seem to be so abnormal. “They are entirely consistent with what AMPs do,” Moir said.

What causes antimicrobial defense to go south toward Alzheimer disease pathology is unknown, but in his talk at AD/PD, Rudy Tanzi, who directs the Genetics and Aging Research unit at MGH, offered genetic hints that might provide reason to investigate further. Genomewide association screens for AD that Tanzi’s group is currently conducting are generating signals from genes involved in the molecular skirmishes that have evolved between host and microbes, as well as from genes whose proteins signal to activate the innate immune system. CD33 is one of four genes recently published to have genomewide significance (Bertram et al., 2008). CD33 plays a role in the innate immune system, and other such genes came up just below the threshold of significance. “These genes kept hitting us over the head that something must be going on with innate immunity,” Tanzi said. “Conceivably, it could be that if you have inherited weak risk alleles in innate immunity components that are less active, the body might compensate by making more Aβ to fight off infections.” Moir, Tanzi, and James Kirby, an infectious disease specialist at Beth Israel Deaconess Hospital in Boston, struck up a three-way collaboration to pursue the idea, and Moir presented the data in Prague.

For his part, Moir had his curiosity piqued by his weekly playtime before Friday beer hour, when he surfs PubMed for interesting papers until he gets bored and goes to grab his brew. “One day I perused AMPs and noticed similarities to Aβ, so I looked, and looked some more for three months. I scoured the literature and now my list of papers on similarities between Aβ and AMPs is 200 citations long,” Moir said.

For comparison, take cathelicidin, aka LL-37, an established human AMP found in leukocytes and macrophages. Its proven arsenal includes killing bacteria, viruses, virus-infected and even cancer cells, as well as attacking the mitochondria of parasites. Moir began his talk by flashing a list of commonalities between LL-37 and Aβ that includes these features: both are small, secreted, pro-inflammatory peptides; both form amyloid fibers, aggregate best in the presence of membranes, and associate with plaque lesions (atherosclerotic plaque in the case of LL-37). Their most potent cytotoxic forms are oligomers; both are fairly protease-resistant, bind apolipoproteins, and activate the innate immune system. “Almost every major property of LL-37 is described in the AD literature as a pathological property of Aβ,” Moir said.

This prompted the scientists to test whether Aβ kills microbes. In Prague, Moir presented results comparing LL-37 and Aβ in standard assays establishing the minimal inhibitory concentration at which each peptide eliminates half of the microbes present. In this experiment, Aβ’s MIC was in the low micromolar range, the same as that of LL-37 (see, e.g., Johansson et al., 1998). Aβ was active in this way on E. coli, staphylococci, Listeria monocytogenes, the Borrelia spirochete, Helicobacter pylorus, Chlamydia pneumoniae, the fungus Candida albicans and other pathogens. Aβ42 is slightly more potent than Aβ40, and both tend to be more bacteriostatic than bacteriocidal, Moir noted. In comparing the potency of Aβ oligomers against neurons and against microbes, Moir claimed a difference of at least one order of magnitude. “Aβ is more bacteriotoxic than neurotoxic,” Moir said, adding that that, too, is consistent with a primary role in innate immunity.

Turning the tables, the scientists then took some of what they knew about Aβ and tried it on LL-37. For one, it is known that certain metals mediate Aβ oligomerization, whereas the role of metals is mostly absent from the AMP literature. In experiments with LL-37, trace amounts of copper mediated formation of dimers and trimers, and it was oligomeric species, not the monomer, that attacked bugs, Moir reported in Prague. For another, Aβ’s ability to generate hydrogen peroxide is known as part of its neurotoxic weaponry. This oxidizing acid, as well as oxygen radicals, is also known to form part of a host’s defense mechanism against microbes but was not considered part of the AMP arsenal. In Prague, Moir showed data suggesting that LL-37 generates hydrogen peroxide, and indeed might deploy it twice against bacterial membranes—first generating H2O2 from membrane phospholipids and then unleashing it on the membrane for further oxidative damage.

One reason why microbiologists may have overlooked Aβ is that most AMPs are cationic peptides, whereas Aβ is anionic. But by now some anionic AMPs are known, and the general view is that both anionic and cationic AMPs have evolved as part of the measures and countermeasures that host and microbe inflict on each other, Moir said.

AMPs are evolutionarily ancient. Organisms as old as horseshoe crabs use them, as do even bacteria in their war against other bacteria. The innate immune system precedes the adaptive immune system. Unlike the adaptive system, which selectively expands responsive B and T cells to mount a bigger response after it re-encounters a known intruder, the innate immune system responds in the same way every time. The innate immune system has been implicated in AD before (e.g., Giunta et al., 2008; Macchioni et al., 2009). For example, some of its signaling pathways through toll-like receptors are beginning to generate a small literature in AD research (e.g., Lotz et al., 2005; Walter et al., 2007; Scholtzova et al., 2009; Tan et al., 2008; Udan et al., 2008). Many prior studies focused on activation of these pathways by Aβ, but toll-like receptors act even upstream of that, whereby bacterial ligands such as lipopolysaccharide (LPS) activate these receptors, which then signal to increase expression of AMPs, Moir said.

This work evokes parallels between the AMP and AD literature. For example, many AMPs work through amyloid formation. They generate crystalline deposits on bacteria that disrupt the membrane, echoing a toxicity mechanism proposed for Aβ and α-synuclein (e.g., Yoshiike et al., 2007; Lashuel et al., 2002; ARF Live Discussion; from the AMP literature, see Jang et al., 2008).

Another parallel from the AMP literature lies in neutrophil extracellular traps (NETs). These are known from horseshoe crabs and spiders, whose own blood-derived AMPs rapidly coagulate a crystalline material around infectious agents to entrap them. It’s basically a proteinaceous net that immobilizes and then kills the pathogen. These NETs occur in human inflammatory reactions against bacterial infection as well (see Brinkmann et al., 2004).

“We think Brad Hyman’s observation that plaques can form very fast is a key one,” Moir said. “You could speculate that amyloid deposition is not just a question of Aβ concentration rising to a threshold level, but that physiologically, it can be an active process to entrap an infection.” This would raise the question of whether inside every plaque lie the remains of a microbe.

In higher organisms, platelets contribute to NETs in blood in cases of extreme bacterial infection (Clark et al., 2007). Platelets are (besides muscle) the leading known source of Aβ outside the brain. There is at present no data to indicate that Aβ plays a role in mopping up pathogens that enter the bloodstream, though it is known that APP in platelets functions in blood clotting. Other scientists have reported that both LL-37 and Aβ42 can attract leukocytes (Le et al., 2002).

In Prague, this talk generated the kind of healthy “show-me-more-data” skepticism that typically greets unpublished results drawing heavily on an outside literature. For one, scientists cautioned that Aβ kills all sorts of cells, so might well kill bacteria, too, without that being an important physiological function. For another, there are no indications that people who have low Aβ levels are prone to infection. One way to test this hypothesis would be to see whether transgenic mice that overexpress Aβ are more resistant to infection, and knockouts more susceptible. LL-37 is clearly important physiologically, as LL-37 knockout mice die from infections. Whether mice lacking Aβ are prone to infection was not immediately clear. BACE1 knockout mice are healthy (e.g., Roberds et al., 2001), though one research group has reported at meetings that BACE1/BACE2 double knockout mice that lack Aβ in all body tissues tend to die young, possibly from infection (see ARF related news story).

Even if this new research stands the test of independent replication, what does it mean for AD? “We really don’t know yet,” Moir said. It may be that a role of Aβ as an AMP and its aggregation in AD are unrelated. At a speculative level, one alternative explanation would be that some cases of AD could result from a persistent CSF infection, or from a transient infection that went away but engendered a permanent Aβ response and its attendant immune modulation. These words sound similar to a long-standing hypothesis by investigators such as Ruth Itzhaki and Brian Balin, who maintain that persistent infection with herpes simplex or Chlamydia, respectively, might be at the root of some cases of AD (see ARF Live Discussion; Wozniak et al., 2009; Balin et al., 2008). “They might be right,” Moir said, “though we would propose a more generic mechanism, not one based on any particular microorganism.”—Gabrielle Strobel.

Man in Prague Playing Hurdy-Gurdy
Called a Ninera in Czech, this medieval instrument has a deep tradition in Eastern Europe. Image credit: Benjamin Wolozin

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Prague: Piecing Together Pathology with PyroGlu

16 Apr 2009

Pyroglutamate, or pyroGlu (pGlu) to the initiated, is an aromatic ring form of glutamate that has been discovered in some fragments of amyloid-β, namely, those missing the first two (Aβ3-x) or 10 (Aβ11-x) amino acids. Growing evidence suggests that pGlu forms of Aβ play a role in the pathology of Alzheimer disease (AD), given that they are more stable and more likely to seed aggregation (see ARF related news story) than the unmodified peptides. But research into pGluAβ has so far found traction among only a few AD researchers. That seems poised to change. “There are now quite a few labs beginning to work on this,” Cynthia Lemere, Brigham and Women’s Hospital, Boston, told ARF. Lemere, who heads one of those labs, recently chaired a pyroGluAβ session at the 9th International Conference AD/PD, held 11-15 March in Prague, Czech Republic. Among the reports that stood out at that session were those showing that other proteins linked to the pathology of dementia undergo the same cyclization process, and that in addition to BACE, there may be other secretases that cleave APP, suggested Lemere. Those mystery secretases might cleave between the second and third amino acids of the Aβ sequence of APP, leaving an N-terminus glutamate that would be vulnerable to cyclization. For her part, Lemere is keeping some hot pyroGlu data under her hat until the International Conference on Alzheimer’s Disease and Related Disorders next July in Vienna, Austria.

The Prague pyroGlu session was sponsored by Probiodrug AG, Halle, Germany, a biotechnology company that has been driving much of the research into this variant of Aβ. Probiodrug’s Stephan Schilling described some biophysical properties of pyroGlu Aβ, showing that at pH 5-7, pGluAβ3-x is less soluble than the full-length peptide. “This reduced solubility is the driving force for aggregation propensity at physiological pH,” said Probiodrug’s Uli Demuth in a post-meeting interview with this reporter. Schilling also reported that ABri and ADan, the amyloidogenic peptides responsible for British and Danish dementia, respectively, undergo a similar N-terminal pyroglutamate cyclization and that this renders these peptides more insoluble and more hydrophobic, as well. “I thought this was amazing, as it suggests that the same mechanism is consistent among these Aβ-like peptides,” said Lemere. In fact, pGluABri and pGluADan are even more insoluble than pGluAβ3-42. “This reflects their relative toxicities and is in keeping with the fact that they [ABri and ADan] are primarily found in the vasculature and are not readily transported,” said Demuth.

Holger Cynis, also from Probiodrug, offered up yet another dementia-relevant substrate for glutaminyl cyclase (QC), the enzyme that catalyzes pGlu formation. Monocyte chemoattractant protein 1 (MCP-1, also known as CCL2) is a major monocyte/glial chemokine that is elevated in AD (see Galimberti et al., 2006) and may mediate the chronic inflammation associated with the disease (see Sokolova et al., 2008). Blocking MCP-1 signalling in the brain suppresses gliosis, Aβ accumulation, and learning impairment in APP/PS1 mice (see Kiyota et al., 2009). Cynis showed that the N-terminus glutamine of MCP-1 can be cyclized and that the resulting pGlu form of the chemokine was the most potent form in a cellular assay of chemotaxis. pGluMCP-1 seemed relevant in an in-vivo model of peritonitis based on injecting thioglycollate to stimulate monocytes, because administering a QC inhibitor 30 to 60 minutes before immune stimulation dramatically reduced monocyte infiltration.

But the story is more complex than that. MCP-1 is not only susceptible to QC but also to proteases that remove peptides from its N-terminus. One of these proteases is dipeptidyl dipeptidase 4 (DP4), which nibbles away the first two amino acids of proteins when proline is in the second position. Cynis showed that DP4 cleavage of the glutamine-proline N-terminus of MCP-1 turns the chemokine from an agonist to an antagonist. Because DP4 does not cleave pGluMCP-1, elevated QC could therefore cause a double whammy, Demuth suggested. By simultaneously stabilizing Aβ and MCP-1, QC might exacerbate both the pathology and neuroinflammatory consequences of Aβ toxicity. “We think pGluAβ may be increasing with age, initiating the death of neurons, causing release of MCP-1, which in turn activates resting microglia that upregulate QC, leading to even more MCP-1,” said Demuth. “QC inhibition would, therefore, hit both sides of that coin.” Support for that idea came from Makoto Higuchi, National Institute of Radiological Sciences, Molecular Imaging Center, Chiba, Japan. In a different session he reported that in AD mouse models, reactive microglia are characterized by massive overexpression of MCP-1.

New animal data support this scenario. Stephan von Horsten, also from Probiodrug, presented data showing that QC is expressed in several nuclei of the rat brain. The enzyme is found in neurons, microglia, and astrocytes in many regions of the brain including the hippocampus, various cortical structures, the striatum, the thalamus, and hypothalamus. Van Horsten also reported that injecting Aβ into the brain caused an increase in QC and in the number of QC-positive microglia (see Schilling et al., 2008). In primary human brain cell cultures, QC is expressed in astrocytes and microglia. In the poster session, Anca Alexandru from Probiodrug’s daughter company Ingenium Pharmaceuticals in Munich showed that an animal model of pGluAβ toxicity fits with the pGluAβ/activated microglia double hit hypothesis. In mice expressing Aβ3-42 directly from the thy1 promoter, highly sensitive ELISAs detect pGluAβ3-42 in the hippocampus after just four weeks of age. At two months, the animals exhibit different severe behavioral and locomotive phenotypes, at three months there is neuron loss accompanied by pGluAβ immunohistochemical staining, microgliosis, and astrogliosis, and by five months no more invading glia are seen “because there is nothing left to clean up,” said Demuth.

Thomas Bayer, University of Goettingen, Germany, also reported on a pGluAβ model, wherein Aβ3-42, having either a glutamate or a glutamine at the 3 position, is fused to mouse pre-pro-thyrotropin releasing hormone. These models generate large amounts of intraneuronal pGluAβ3-42 and by eight weeks of age have extensive neurological impairment that seems to correlate with Purkinje cell loss. Bayer also reported on an APP/presenilin-1 knock-in mouse, which develops neuron loss, axonopathy, and synaptic and learning deficits that all seem to correlate with intraneuronal aggregation of Aβx-42 (see ARF related news story). Interestingly, pGluAβ is elevated about 40-fold in the brains of these animals compared to controls. Those findings support the idea that intraneuronal Aβ, and pGluAβ in particular, may be a major player in AD pathology.

Probiodrug hopes that inhibition of QC will be a strategy for treating AD. In Prague, Steffen Rossner, University of Leipzig, Germany, reviewed some preclinical data published toward that goal (see ARF related news story). Briefly, in this study inhibiting QC in two different mouse models reduced pGluAβ3-42, total Aβ, and overall plaque burden, and improved performance in learning and memory tasks. QC inhibition reduced gliosis, which could be due, in part, to reining in MCP-1. Demuth told ARF that QC inhibition can prevent atherosclerotic plaques, which are driven by inflammatory responses, in mice fed a high cholesterol/fat diet. The Austrian biotech company AFFiRiS, betting on pGluAβ being a player in AD, has started a vaccine program to target these peptides.

There are still questions lingering over the pGluAβ peptides. Above all, it’s unclear how the N-terminus gets truncated to begin with and how these pGlu peptides might be expunged from the brain. Aβ3-42 could be formed in two ways. Either the responsible protease truncates full-length Aβ peptide N-terminally, or it cleaves APP between position 2 and 3. That implies that some non-BACE secretase is at work. “My feeling is that Aβ3-x is generated by an unknown protease that removes the two N-terminal amino acids of Aβ after the latter is formed, so that the formation of Aβ3-x depends on prior BACE1 cleavage,” wrote Bob Vassar, Northwestern University, Evanston, Illinois, in an e-mail to ARF. Vassar did not attend the pyroGluAβ session in Prague, but his lab has done seminal work on BACE. “I think pyroGluAβ is potentially important in AD because it's very stable, but I don't think it's BACE1 independent,” he wrote.

Demuth has been coy about identifying the mystery protease. At AD/PD he presented indirect evidence to suggest that secretases other than BACE may indeed be involved in pGluAβ formation. He reported that in BACE1/2 knockout fibroblasts expressing wild-type APP, the same amount of pGluAβ is formed as in BACE-expressing fibroblasts. “We believe there are many different β site activities,” Demuth said. “We have studied that in different cell systems, including primary cortical neurons, and we see about one-half of Aβ is formed via the BACE pathway. The other half is formed by different protease(s), among them being the proteolytic pathway that generates pGluAβ,” said Demuth.

“This is a very hot topic,” suggested Lemere. “There is definitely a lot of work going on in multiple labs right now investigating whether there are other secretases involved in pre-cleavages or cleavage of Aβ at the -1, -2, +2, +3 positions, etc., and it may be that enzymes are responsible for those alternative cleavage sites,” she said.

Last but not least, what happens to pGluAβ once it is formed? In his AD/PD presentation, Takaomi Saido of the RIKEN, Wako, Japan, reported that pGluAβ is very much resistant to in vivo degradation. The half-life of pGluAβ3-42 in the brain was approximately five times longer than full-length Aβ42. In contrast, truncated Aβ species other than pGluAβ3-42, such as Aβ3-42 and Aβ4-42, were more susceptible to in vivo degradation than Aβ42. This may account for the selective deposition of pGluAβ in aging human brains, suggested Saido. He also demonstrated, by immunohistochemistry and mass spectrometry, that neprilysin deficiency resulted in elevated deposition of pGluAβ in APP-Tg mice, indicating a connection between the aging-associated decline of neprilysin activity and accumulation of pGluAβ in the brain. The underlying mechanism remains yet to be identified. Strikingly, Saido and colleagues found that QC is upregulated about fivefold in the APP/NEP-/- animals. This is relevant to earlier work by Demuth’s group showing upregulation of QC in brain tissue of AD patients but not of age-matched controls (see Schilling et al., 2008). Whether the same driving force is responsible for elevated QC in aging humans and NEP-negative mice remains to be seen.—Tom Fagan.

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References

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Galimberti D, Schoonenboom N, Scheltens P, Fenoglio C, Bouwman F, Venturelli E, Guidi I, Blankenstein MA, Bresolin N, Scarpini E. Intrathecal chemokine synthesis in mild cognitive impairment and Alzheimer disease. Arch Neurol. 2006 Apr;63(4):538-43. PubMed.
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External Citations

Spectrum of Neurodegeneration Comes to the Fore

28 May 2009

Sometimes in science, a concept lingers unattended in the collective back of researchers’ minds for years; they sense there’s something important to it but aren’t ready to grasp it head-on. Then something changes and, voila, the concept moves front and center. Such was the case with the issue of overlap in the major neurodegenerative diseases at the 9th International Conference AD/PD held 11-15 March in the Czech capital city of Prague. Previous AD/PD conferences had reflected the de-facto separation in the daily work of most scientists and clinicians in this field. Even though they spent days under the same roof, by and large the movement disorder people went to one meeting and the dementia people to another. But Prague was different. Rather than being treated as an inconvenient side issue that blurred boundaries, the extensive and multitudinous overlap between Alzheimer and Parkinson diseases (and also the frontotemporal dementias) was the focus of presentations and hallway discussion. Much of the buzz about this topic had spilled over into Prague from a preceding workshop in the former Royal German city of Kassel that had focused on dementia with Lewy bodies (DLB) and Parkinson disease dementia (PDD). These are two underappreciated conditions that occupy a large area of overlap between AD and PD. From 8-10 March, co-organizer Brit Mollenhauer of the Paracelsus-Elena-Klinik in Kassel had convened a group of investigators around the goals of sharing the latest insight and hammering out a research agenda to speed progress and fight for recognition of these betwixt diseases.

Once the overlap between two neurodegenerative diseases, not their differences, becomes the center of attention, the story changes in many ways, scientists said. Individual cases are comfortably seen as falling on a spectrum rather than having to fit into this box or that, and the view of the diagnosing physician changes such that (s)he expects and accommodates large numbers of mixed cases. Perhaps most importantly, the search for protein-based biomarkers—from fluid biochemistry to imaging—to tease out which underlying proteins drive a given person’s disease, assumes paramount importance.

Traditionally, neurodegenerative diseases have been classified clinically, as physicians grouped them into boxes based on the preponderance of signs their patient presented—movement abnormalities in PD, cognitive deficits in AD at its simplest. Separately, pathologists described postmortem brain abnormalities and tried to match them up with symptoms. That clinico-pathological pairing is descriptive, and it has been refined in recent years. But in reality, the pathology, seen years after diagnosis, often poorly matches the clinical diagnosis a patient had received. Or it even calls the diagnosis into question when, for example, a patient diagnosed as having AD turns out to have had extensive α-synuclein but little tau pathology in the brain.

“There has been a conceptual shift in neurodegeneration research. Until recently the focus was on factors that distinguish between these disorders. But now we recognize that there is extensive overlap at the pathological level and the clinical level, which does not fully match the genetics. So it is difficult to distinguish cleanly by means of clinical, pathological, or genetic determination alone,” said Kristel Sleegers of VIB in Belgium.

As science advances, the diagnosis of neurodegenerative diseases beyond AD itself will become increasingly molecular in an effort to pin down the pathogenic proteins that drive an individual person’s disease rather than focus primarily on the symptoms. “In time, we will shift away from relying on clinical categorization to make diagnoses,” said James Galvin of Washington University, St. Louis. “We will make protein diagnoses. The way to get there is to understand underlying pathways and to develop a range of biomarkers.”

The need for protein markers (and eventually also RNA- and lipid-based markers) as sorting tools is evident from even a cursory look at how varied these diseases can be. For example, scientists are realizing that what looks like a single clinical entity has multiple causes. To quote but one emerging example, new studies of Parkinson disease patients are fingering the gene for Gaucher disease, as well as a high-expression variant of the protein tau (see Part 9, Part 2 in this series). Vice versa, a single pathogenic mutation, for example, in the gene progranulin, has been reported to manifest itself in the form of frontotemporal dementia, Parkinson’s, or even Alzheimer’s in affected members of one and the same family.

Confused already? Read on; there’s more. DLB and PDD define a spectrum going from AD to PD that hinges on the specifics of aggregation of Aβ, tau, and α-synuclein, but it is not the only spectrum at play. There is also a spectrum going from frontotemporal dementias to amyotrophic lateral sclerosis that hinges on loss of progranulin protein and accumulation of ubiquitin and TAR DNA-binding protein 43 (TDP43). Deposits of the latter have been reported in a significant fraction of AD cases, even, though whether they are mechanistically important to disease is entirely unclear at this point. And, of course, it matters greatly where a pathologic protein acts. For example, of the wide range of disorders blamed on α-synuclein—the Lewy body diseases—one particularly severe one called multiple system atrophy stands out by exhibiting this pathology primarily in oligodendroglia, not neurons. Yet another spectrum talks of tauopathy in parkinsonism (see Part 2 of this series.) The number of invoked spectra conjure up a mental image less of a linear continuum between, say, blue and green, but more of a color wheel. Scientists readily agree that there are many more examples for heterogeneity at the clinical, pathological, and genetic levels.

“People are talking in terms of spectra now because the pure forms of these diseases are less common than we used to think, and the overlap is incredibly large,” said Galvin. “Once you focus on patients in the overlap, you see that they are different from those with the pure forms of disease. That is why molecular diagnoses are going to be critical for this field.”

Molecular diagnoses will require fluid or imaging markers based on individual proteins that drive disease either alone or in various combinations. The range of candidates is expanding well beyond the known ones such as amyloid-β, tau, to now include α-synuclein (Part 6 of this series), progranulin (Part 7), TDP43, glucocerebrosidase (see Part 9), and brain imaging based on neurotransmitter transporters (see Part 5 of this series. Such tests are being aggressively pursued in different labs.

Besides offering a more precise diagnosis, such tests may help scientists define what molecular interactions underlie yet another phenomenon scientists emphasized both in Kassel and in Prague—namely that when two of these proteins go awry in one person, they tend to heat up the pathogenic process and worsen the resulting clinical disease in a given person. “There’s an emerging realization that whenever two pathologies occur together, they accelerate disease,” said Michael Schlossmacher of the University of Ottawa, Canada. This, in turn, has created interest in studying possible links between the underlying proteins at the monomeric and oligomeric level (see Part 4 of this series).

The esteemed reader trying to keep track of these blurring boundaries might take solace in remembering that this added layer of complexity comes on top of a much simpler, underlying rule that has found wide acceptance across the field of neurodegeneration. It is that for the established, major proteins causing neurodegeneration when they aggregate —Aβ/amyloidoses, α-synuclein/synucleinopathies, tau/tauopathies, prp/prion diseases—mutations or duplication of the gene cause familial forms, whereas overproduction alleles raise the risk of sporadic forms. “This blindingly simple categorization is true for all of these diseases. The more you make of these proteins, the earlier you get the disease,” John Hardy of University College, London, UK, said in Prague. And as a possible yang to this yin, the opposite trend is just beginning to emerge for progranulin and perhaps even the newest neurodegenerative disease protein, glucocerebrosidase. There, early indications are that disease risk goes up the less a person makes of the protein.—Gabrielle Strobel.

This story leads a nine-part series. See also Part 2, Part 3, Part 4, Part 5, Part 6, Part 7, Part 8, Part 9.

Wilhelmshoehe, Europe's biggest hillside park with 600 species of trees, as well as castles and cascading waterfalls, overlooks Kassel, the old German city hosting a recent workshop on DLB and PDD. Image credit: Paracelsus-Elena-Clinic, Kassel

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Et tu, Brute? Parkinson’s GWAS Fingers Tau Next to α-Synuclein

29 May 2009

At the 9th International Conference AD/PD, held this past March in Prague, Thomas Gasser of the Hertie Institute in Tuebingen, Germany, presented data that illustrated two emerging concepts in neurodegenerative disease research (see Part 1 of this series). First, the same gene can cause disease in various guises—as an inherited mutation in some familial cases or as a risk variant in some sporadic cases. Second, the overlap between related neurodegenerative diseases is tremendously large and varied. The overlap cuts across the clinical, pathological, and genetics levels, and tau rears its head again and again.

Gasser presented the results of the largest genomewide association study (GWAS) performed to date on Parkinson disease (PD) patient samples. In PD, geneticists in the past decade have assembled a list of some 15 genetic loci. Even so, the large majority of the genetic variance underlying the total burden of PD remains unexplained. The best-understood gene, α-synuclein, gives rise to rare familial PD when mutated, duplicated, or triplicated. However, at the level of pathology, its Lewy body signature is much more widespread than that (Spillantini et al., 1997). Indeed, even beyond PD, α-synuclein pathology shows up in a range of other α-synucleinopathies (see Part 8 in this series). The genetic contribution of α-synuclein alleles to that larger burden of neurodegenerative disease is largely unknown.

The present GWAS represents a collaboration of an international PD Genetics consortium with Andrew Singleton at the National Institutes of Health in Bethesda, Maryland, and additional groups elsewhere, Gasser said. The scientists studied 463,000 quality-controlled SNPs per sample—a huge number as such studies go—in some 1,700 quality-controlled (qc’d) cases and 4,000 qc’d controls from mostly Caucasian U.S. and German samples in a first stage, and then analyzed the 384 top SNPs from this stage in a second sample of 3,500 cases and 4,200 controls. “This gave us good power to detect common risk factors conferring modest effects,” Gasser said.

Which genes survived statistical correction to remain above the Bonferroni line? Only two did—not LRRK2, not parkin, PINK1, or DJ1. α-synuclein and tau stood out in both stages of the GWAS, Gasser reported. For α-synuclein the result was somewhat expected. It further cemented the relationship in certain key genes between pathogenic mutations, duplication, or triplication causing familial and risk alleles promoting sporadic forms of PD. In contrast, tau’s appearance at first glance may seem an astonishing betrayal by a protein implicated in AD and FTD, not primarily PD. But it shouldn’t, noted other researchers. For example, in some cases of PD, tau pathology is readily apparent, said Michael Schlossmacher of the University of Ottawa, Canada. Suspicions about tau playing a role in the pathogenesis of PD arose years ago (e.g., Golbe et al., 2001; Maraganore, 2001). Since then, smaller genetic studies have implicated tau in PD in such a way that a low-expression variant appears to be mildly protective (see tau on PDGene). In the present GWAS, tau’s genetic effect may have been mediated through mRNA expression levels, Gasser said. And the idea is catching on. German researchers recently proposed a spectrum of tauopathy with parkinsonism (Ludolph et al., 2009), and yet other scientists have blamed certain tau variants for the dementia that develops in many patients with advanced PD (Goris et al., 2007). Greetings from the overlap—again.

“In most GWASs of these neurodegenerative diseases, tau comes up. It is not clear what it does, but one thought is that here it could act as a switch to augment α-synuclein pathology,” commented Douglas Galasko of the University of San Diego, California.

Indeed an old finding from familial AD brought up this connection between tau and α-synuclein, when researchers observed that people who had inherited an autosomal-dominant presenilin or APP mutation at autopsy turn out, besides the expected plaques and tangles, to also have had Lewy body pathology in their brains (Lippa et al., 1998). Researchers increasingly agree that APP or presenilin lies upstream of tau in the AD disease pathway, but tau then appears to somehow fire up α-synuclein, as well. What exactly goes on between the two, or whether the pathways are separate, is anyone’s guess at this point, scientists said.

Beyond pointing an accusing finger at tau, the new GWAS presented by Gasser confirms the association of α-synuclein variants and sporadic PD found in previous, smaller studies. The odds ratio for α-synuclein computes to 1.4 for carriers of one risk allele and to 1.9 for carriers of two; together this could account for some 9 percent of the population risk for PD, Gasser said in his talk. The idea is that certain risk alleles increase α-synuclein protein levels. How that might happen, however, remains unclear as the mechanism appears to be more complicated than simple differences in α-synuclein mRNA levels, according to ongoing research in the laboratory of John Hardy at University College, London.

A flip side to this overlap between neurodegenerative diseases (i.e., one clinical entity—different genes) is that one gene can give rise to different clinical pictures. The new GWAS sheds light on this, too. Consider multiple system atrophy (MSA). People with this severe and mysterious disease deteriorate rapidly from parkinsonism or ataxia (or both), and from concomitant failure of their autonomic nervous system. Their brains show α-synuclein pathology mainly in glial cells (Wenning et al., 2008). MSA appears entirely sporadic, as no familial cases have been described as yet. But it has enough in common with PD that Gasser and colleagues decided to check whether any of the 384 top SNPs from the PD GWAS were associated with MSA. This second genetic study, as well, was large for such a rare disease, first testing the SNPs in 413 cases and nearly 4,000 controls and then replicating the top 10 SNPs from that stage in a second, independent cohort. Fifteen different institutions in Europe and the U.S. contributed samples to this study, Gasser noted. α-synuclein emerged as the only MSA risk gene to survive replication and statistical correction. It also stood through further replication in a third sample of 100 pathology-confirmed cases from the Institute of Neurology in London, UK. For MSA, the odds ratio of carrying a homozygous α-synuclein risk allele may be as high as 6.2, Gasser showed (Scholz et al., 2009).

Overall, then, this GWAS drove home the message that α-synuclein alleles that lead to higher expression or protein levels can increase a person’s risk for PD and at least one other α-synucleinopathy, MSA. Tau, too, plays a genetic role in Lewy body diseases. Gasser estimated that taken together, the genes for α-synuclein and tau may account for a fifth of Parkinson disease cases.

Questions after his talk revolved around the notion of whether two pathologies, when they occur together, might fire each other up to accelerate disease. The GWAS does not directly address this because the tau and α-synuclein risk variants were independent of each other. But the question deserves study, Gasser said. To many neurogeneticists, tau’s hand on the tiller of Lewy body diseases may come as no surprise. Tau has been placed at the center of another neurodegenerative disease spectrum before, namely that from AD to FTD (Dermaut et al., 2005). And just this month, British researchers reported having spotted it even further afield, in an aggressive form of multiple sclerosis (Anderson et al., 2009).—Gabrielle Strobel

This is Part 2 of a nine-part series. See also Part 1, Part 3, Part 4, Part 5, Part 6, Part 7, Part 8, Part 9.

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References

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Spillantini MG, Schmidt ML, Lee VM, Trojanowski JQ, Jakes R, Goedert M. Alpha-synuclein in Lewy bodies. Nature. 1997 Aug 28;388(6645):839-40. PubMed.
Golbe LI, Lazzarini AM, Spychala JR, Johnson WG, Stenroos ES, Mark MH, Sage JI. The tau A0 allele in Parkinson's disease. Mov Disord. 2001 May;16(3):442-7. PubMed.
Maraganore DM, Hernandez DG, Singleton AB, Farrer MJ, McDonnell SK, Hutton ML, Hardy JA, Rocca WA. Case-Control study of the extended tau gene haplotype in Parkinson's disease. Ann Neurol. 2001 Nov;50(5):658-61. PubMed.
Ludolph AC, Kassubek J, Landwehrmeyer BG, Mandelkow E, Mandelkow EM, Burn DJ, Caparros-Lefebvre D, Frey KA, de Yebenes JG, Gasser T, Heutink P, Höglinger G, Jamrozik Z, Jellinger KA, Kazantsev A, Kretzschmar H, Lang AE, Litvan I, Lucas JJ, McGeer PL, Melquist S, Oertel W, Otto M, Paviour D, Reum T, Saint-Raymond A, Steele JC, Tolnay M, Tumani H, van Swieten JC, Vanier MT, Vonsattel JP, Wagner S, Wszolek ZK, . Tauopathies with parkinsonism: clinical spectrum, neuropathologic basis, biological markers, and treatment options. Eur J Neurol. 2009 Mar;16(3):297-309. PubMed.
Goris A, Williams-Gray CH, Clark GR, Foltynie T, Lewis SJ, Brown J, Ban M, Spillantini MG, Compston A, Burn DJ, Chinnery PF, Barker RA, Sawcer SJ. Tau and alpha-synuclein in susceptibility to, and dementia in, Parkinson's disease. Ann Neurol. 2007 Aug;62(2):145-53. PubMed.
Lippa CF, Fujiwara H, Mann DM, Giasson B, Baba M, Schmidt ML, Nee LE, O'Connell B, Pollen DA, St George-Hyslop P, Ghetti B, Nochlin D, Bird TD, Cairns NJ, Lee VM, Iwatsubo T, Trojanowski JQ. Lewy bodies contain altered alpha-synuclein in brains of many familial Alzheimer's disease patients with mutations in presenilin and amyloid precursor protein genes. Am J Pathol. 1998 Nov;153(5):1365-70. PubMed.
Wenning GK, Stefanova N, Jellinger KA, Poewe W, Schlossmacher MG. Multiple system atrophy: a primary oligodendrogliopathy. Ann Neurol. 2008 Sep;64(3):239-46. PubMed.
Scholz SW, Houlden H, Schulte C, Sharma M, Li A, Berg D, Melchers A, Paudel R, Gibbs JR, Simon-Sanchez J, Paisan-Ruiz C, Bras J, Ding J, Chen H, Traynor BJ, Arepalli S, Zonozi RR, Revesz T, Holton J, Wood N, Lees A, Oertel W, Wüllner U, Goldwurm S, Pellecchia MT, Illig T, Riess O, Fernandez HH, Rodriguez RL, Okun MS, Poewe W, Wenning GK, Hardy JA, Singleton AB, Del Sorbo F, Schneider S, Bhatia KP, Gasser T. SNCA variants are associated with increased risk for multiple system atrophy. Ann Neurol. 2009 May;65(5):610-4. PubMed.
Dermaut B, Kumar-Singh S, Rademakers R, Theuns J, Cruts M, Van Broeckhoven C. Tau is central in the genetic Alzheimer-frontotemporal dementia spectrum. Trends Genet. 2005 Dec;21(12):664-72. PubMed.

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Neither Fish Nor Fowl—Dementia With Lewy Bodies Often Missed

01 Jun 2009

Perhaps the biggest, and quintessential, representative of a spectrum neurodegenerative disease is dementia with Lewy bodies (DLB). By some counts, this disease is the second most common form of dementia after Alzheimer disease (AD), with patient estimates for its various forms ranging between one and two million in the U.S. (Aarsland et al., 2008; Weisman and McKeith 2007). All the same, DLB has struggled for recognition and research dollars, being squeezed uncomfortably between its two large neighbors AD and Parkinson disease (PD). “DLB research is an unappreciated field,” said Brit Mollenhauer of the Paracelsus-Elena-Klinik in Kassel, Germany.

DLB is a double whammy of a disease. People with DLB have behavioral and memory problems as in AD and, to a varying extent, also suffer motor symptoms as seen in PD. However, the cognitive symptoms of people with DLB tend to fluctuate frequently, their motor symptoms are milder than in PD, and DLB patients often have vivid visual hallucinations and particular visuospatial deficits. In short, DLB is neither AD nor PD, and yet defining its distinct identity has been a challenge. At the 9th International Conference AD/PD held last March in Prague, ample discussion about DLB resonated from an immediately preceding workshop on this disease and its cousin, Parkinson disease dementia (PDD). Co-organized by Mollenhauer and Richard Dodel, the workshop tried to put DLB more firmly on the research map (see Part 1 of this series).

“Scientists who study DLB think it is a very important disease,” said James Galvin of Washington University, St. Louis. “As a set of independent groups, we and others have worked to increase its face time in the dementia world. We fight a battle because given the limited time and resources funders and reviewers have available to cover related conditions, DLB tends to get the short end of the stick.”

Why is that? Part of the reason is that carving distinct disease categories out of a continuum of symptoms and pathologies is inherently arbitrary. Part of it is that multiple labels being advanced by different investigators for multiple similar variants have not helped the branding. In Prague, several scientists noted that if its awkward name was part of DLB’s identity problem, one solution might be to name it after Kenji Kosaka of Houyuu Hospital in Yokohama, Japan (Kosaka et al., 1980). “Kosaka himself called it a different name, but he really described the clinico-pathological entity that we nowadays diagnose as DLB,” said Michael Schlossmacher of Ottawa University, Canada. “Alzheimer disease, Parkinson disease, Kosaka disease would sound consistent and recognizable to me.”

The Kassel workshop was the latest in a series of small international meetings by a consortium of groups interested in DLB and PDD. In 1995, researchers led by Ian McKeith of Newcastle General Hospital met in Newcastle-upon-Tyne, UK, to hammer out consensus diagnostic criteria (McKeith et al., 1996). This spurred diagnosis in specialty settings and provided a basis for gathering incidence and prevalence data there (Zaccai et al. 2005). But the consensus criteria have not widely penetrated community geriatric, neurology, or primary care settings where many patients are still seen, and both the rate and accuracy of DLB diagnosis remain low. “We are very bad at diagnosing DLB. Up to half of cases diagnosed as DLB turn out at autopsy to have had AD. Misdiagnosis of DLB as AD occurs, as well,” said David Brooks of Imperial College, London.

This has serious consequences. The U.S. Food and Drug Administration, for example, does not formally recognize DLB as a distinct disorder. The agency has a point, Galvin concedes. “They ask: Is it AD? Is it PD? What exactly is it? They ask: Do doctors recognize DLB as separate? No? Then how can you run drug trials, and how can you get doctors to prescribe a future DLB drug?” Galvin said.

With their series of DLB/PDD workshops, McKeith’s and other groups aim to forge a common research agenda that can move their nascent field forward in a concerted way. Subsequent to Newcastle, workshops in the Dutch city of Amsterdam and Yokohama, Japan, continued the effort; and this year’s gathering in Kassel was to lead up to an official centenary workshop in 2012 that will celebrate Frederick Henry Lewey’s first description of Lewy bodies in 1912. (Born in Berlin as Fritz Heinrich Lewy, this German Jewish neurologist-cum-pathologist in his early years worked with Emil Kraepelin and Alois Alzheimer (see Centennial Alzheimer story), but was forced in 1933 emigrate to England and then the U.S.) This year’s workshop designated working groups for biomarkers and for clinical trials, said Mollenhauer. It also included representatives from national DLB societies and patient groups in an effort to help these lay groups beef up their operations such that they can become larger funders of research and lobby for federal funding and recognition, akin to what the Alzheimer’s Association has accomplished for its disease.

Below are some of the main problems, and points of consensus, about DLB from the Kassel and Prague meetings. Part of the reason why more research groups have not taken up focused study of DLB is its complexity. DLB is marked by overlap with AD and PD on the clinical level and on the postmortem pathology level. But clinic and pathology do not match up to a clean picture, leaving the scientist to juggle a welter of descriptive facts that for many fail to “gel” into a tangible entity. Eventually, the solution to this problem will come with new biomarker-driven diagnoses (see Part 5 and Part 6 of this series), but even in the meantime, clinico-pathological correlations have come a long way, the Kassel workshop made clear.

Clinicians agree that people with mixed pathologies suffer faster and more severe disease. Pure Lewy body pathology exists in 10 to 20 percent of cases with clinical DLB, but the majority of patients also have amyloid pathology and many have tangles to varying degrees, as well. Some even have aggregates of TDP43, though whether that is functionally important is not known yet (Arai et al., 2009; Nakashima-Nasuda et al., 2007). It is beyond dispute, however, that mixed pathologies compound each other. Several groups have found that when they looked at postmortem pathology and compared the clinical and cognitive course of the respective patients during their lives, the mixed cases always performed more poorly and progressed faster. Time to nursing home placement, time to death, visuospatial deterioration—whatever the outcome, the mixed cases fared worse. “Pure and mixed clearly are different diseases,” Galvin said.

In the year 2005, the 1996 DLB consensus criteria were revised to focus on the spectrum of Lewy body disorders and to explain more clearly the links between symptoms and pathology. The clinical aspects that set DLB apart from AD, for example, were ascribed to α-synuclein pathology. In a nutshell, this is what the criteria said: DLB and AD share amyloid pathology; people with DLB have α-synuclein pathology, as well, but generally few neurofibrillary tangles. The more tangles a person has, the more their clinical picture overlaps with AD; the fewer tangles they have, the more it diverges from AD.

In the past four years, several groups have further sharpened the cognitive profile of DLB. The goal is to separate DLB not just from AD but also from PDD, where a patient first has Parkinson disease for some years and then develops dementing symptoms. For example, David Salmon of the University of California, San Diego, showed in Kassel that despite some general similarities between DLB and PDD, PDD is marked by deficits in psychomotor speed and attention, which probably arise as α-synuclein pathology spreads from the brainstem via limbic structures and across the cortex. As long as α-synuclein pathology in the cortex remains mild, PD patients tend to stay cognitively intact (Jellinger, 2009). DLB has in common with AD verbal memory deficits driven by these diseases’ shared amyloid and tau pathologies. That is another point of distinction from PDD (Filoteo et al., 2009). But DLB differs from AD by showing pronounced deficits in visuospatial and executive function that Salmon attributes to a unique combination of cortical amyloid and α-synuclein pathology. These differences are useful early on in disease; in late stages the clinical picture of these diseases increasingly merges. Overall, visuospatial tests seemed the most useful for picking out patients with DLB, and these tended to be the people most likely to deteriorate quickly (Hamilton et al., 2008). They also tended to be the ones who suffered visual hallucinations. Beyond these means, disentangling in more detail which clinical features arise from AD pathology and which ones from α-synuclein pathology will require α-synuclein-based and AD pathology-based biomarkers (see also Lippa et al., 2007).

For his part, Galvin and colleagues recently developed cognitive profiles that distinguish AD from healthy brain aging (Johnson et al., 2008). Compared against these profiles, a group of DLB patients performed quite differently from AD, as well. Combining these cognitive data with clinical and amyloid imaging data, Galvin has devised a clinical risk score that detects LBD. “This gives us a good separation. We find that some people who were clinically diagnosed with AD turn out to probably have DLB,” Galvin said. In the laboratory, his group is working on cerebrospinal α-synuclein detection to support this prediction. The goal is to be able eventually to define preclinical DLB. This would work such that a person who is positive for brain amyloid by PET imaging but is cognitively normal could receive CSF biochemistry testing for Aβ/tau and for α-synuclein to determine whether (s)he will likely go on to develop AD or DLB. “We have great confidence in predicting AD based on the PIB/CSF Aβ-tau combination. We want to achieve the same confidence to predict DLB,” Galvin said. For more on that, see Part 6 of this series.—Gabrielle Strobel.

This is Part 3 of a nine-part series. See also Part 1, Part 2, Part 4, Part 5, Part 6, Part 7, Part 8, Part 9.

References

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External Citations

Like DLB, Like AD—Do Oligomers Stir Up the Trouble?

02 Jun 2009

Current work on distinguishing Alzheimer disease from its cousin dementia with Lewy bodies (DLB, see Part 3 of this series) has underscored one intriguing similarity between the two. In DLB, researchers increasingly note that many people, indeed up to half in some patient series, remain neurologically intact despite having abundant Lewy body pathology in their brains. In AD, florid amyloid pathology in the brains of people who died cognitively normal has for years sustained doubt about the amyloid hypothesis. In DLB now as formerly in AD, the pathologic observation raises questions about whether Lewy bodies are toxic or even relatively protective compared with even more toxic oligomeric aggregates of α-synuclein that remain invisible to the stains typically used on brain slices.

At the Kassel workshop preceding the 9th International Conference AD/PD, Laura Parkkinen of the Institute of Neurology, London, UK, broached this issue in a clinico-pathological talk. Similarly, Walther Schulz-Schaeffer of the University of Goettingen, Germany, proposed that not Lewy bodies, but smaller α-synuclein aggregates at synapses, are the real culprits (Kramer and Schulz-Schaeffer et al., 2007; Kramer et al., 2008). And at AD/PD in Prague, Maria-Grazia Spillantini of Cambridge University, UK, previewed data from a new mouse model of α-synucleinopathy that pinned early pathogenic changes on mislocalization of monomeric α-synuclein within presynaptic terminals (see also Watson et al., 2009). Spillantini proposed that the neurons seen laden with Lewy bodies in autopsy tissue might represent those cells that were the latest to have gotten sick, i.e., that have withstood a disease process driven by smaller assemblies.

Mice are also the model of choice to try to understand whether different pathogenic proteins interact, perhaps as oligomers, before they form their signature microscopic deposits. Eliezer Masliah of the University of California, San Diego, has for some time explored molecular interactions between Aβ and α-synuclein, showing first that Aβ potentiates the deposition of α-synuclein in transgenic mice (Masliah et al., 2001) and more recently that these two proteins can form mixed ring-like oligomers in membranes (Tsigelny et al., 2008 on ARF related news story). In Kassel and in Prague, Masliah expanded on the theme. He introduced several different unpublished mouse systems that combine transgenic lines and lentiviral injection. Together, these build a body of data suggesting that Aβ42 promotes α-synuclein aggregation, worsens learning deficits, and can drive neurodegeneration in these mixed models. This happens regardless of whether APP is added to an α-synuclein transgenic background or α-synuclein is added to an APP-transgenic background.

Most likely, Aβ is upstream of α-synuclein, researchers agreed. This creates a parallel with older Alzheimer’s research placing Aβ upstream of tau, and it puts α-synuclein and tau on a par in a sense. Expressed in mice, the mutant human tau that causes frontotemporal dementia leads to tangles and a behavioral phenotype mostly in the spinal cord (Lewis et al., 2000), but when these mice cross-breed with APP transgenic mice, the Aβ in the resulting offspring greatly amplifies tau pathology in the cortex (Lewis et al., 2001; also Goetz et al., 2001). The idea is that, similarly, the co-occurrence of Aβ might help α-synuclein pathology spread in the human brain.

“The idea of one pathology augmenting another is accepted in AD, and now comes up in DLB, as well,” noted John Hardy of University College, London, UK. Other scientists agreed that amyloid pathology probably deposits first in people, before inciting either tau or α-synuclein pathology. More than two pathogenic proteins can be at play, as some scientists suspect tau heating up α-synuclein pathology downstream of amyloid. “In the mixed pathologies, we know that amyloid deposits first. Then there occurs some unknown step that we need to understand much better,” said Galvin.

This point drew wide notice in Prague. “Broadly, the idea that one of those proteins can influence aggregation of another is gaining prominence,” said Charles Glabe of University of California, Irvine, who mentioned collaborative work with a German group indicating that therapeutic removal of amyloid can draw down α-synuclein inside cells. “The big question is how that happens, whether directly at the membrane or through activating autophagy.”

Interaction between Aβ and α-synuclein might imply that upstream (read anti-amyloid) therapies might benefit downstream α-synuclein pathology (read DLB patients), as well. Masliah showed data suggesting that anti-Aβ immunotherapies treat synucleinopathy and attendant functional deficits quite nicely in transgenic mice. Alas, the known trials and tribulations of translating mouse treatments to humans apply. Other scientists cautioned that diseases marked in large part by pathologies downstream of Aβ amyloid might at some point become independent of that amyloid once disease is established, such that removing the initial offender no longer helps the patient very much because it leaves in place an active tauopathy or synucleinopathy.

Antibodies against α-synuclein are under construction in Masliah’s laboratory. In Prague, Brian Spencer in Masliah’s lab presented a poster showing a lentivirus single-chain antibody against α-synuclein oligomers. When injected into the brain of α-synuclein transgenic mice, the antibody rescued neurodegeneration in these animals. The mechanism, Masliah believes, is not so much microglial clearance but activation of the autophagy pathway of protein degradation. This, if it could be revved up safely, might just offer a new therapy development avenue to pursue against both AD and DLB.—Gabrielle Strobel.

This is Part 4 of a nine-part series. See also Part 1, Part 2, Part 3, Part 5, Part 6, Part 7, Part 8, Part 9.

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References

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Paper Citations

Kramer ML, Schulz-Schaeffer WJ. Presynaptic alpha-synuclein aggregates, not Lewy bodies, cause neurodegeneration in dementia with Lewy bodies. J Neurosci. 2007 Feb 7;27(6):1405-10. PubMed.
Kramer ML, Behrens C, Schulz-Schaeffer WJ. Selective detection, quantification, and subcellular location of alpha-synuclein aggregates with a protein aggregate filtration assay. Biotechniques. 2008 Mar;44(3):403-11. PubMed.
Masliah E, Rockenstein E, Veinbergs I, Sagara Y, Mallory M, Hashimoto M, Mucke L. beta-amyloid peptides enhance alpha-synuclein accumulation and neuronal deficits in a transgenic mouse model linking Alzheimer's disease and Parkinson's disease. Proc Natl Acad Sci U S A. 2001 Oct 9;98(21):12245-50. Epub 2001 Sep 25 PubMed.
Tsigelny IF, Crews L, Desplats P, Shaked GM, Sharikov Y, Mizuno H, Spencer B, Rockenstein E, Trejo M, Platoshyn O, Yuan JX, Masliah E. Mechanisms of hybrid oligomer formation in the pathogenesis of combined Alzheimer's and Parkinson's diseases. PLoS One. 2008;3(9):e3135. PubMed.
Lewis J, McGowan E, Rockwood J, Melrose H, Nacharaju P, Van Slegtenhorst M, Gwinn-Hardy K, Paul Murphy M, Baker M, Yu X, Duff K, Hardy J, Corral A, Lin WL, Yen SH, Dickson DW, Davies P, Hutton M. Neurofibrillary tangles, amyotrophy and progressive motor disturbance in mice expressing mutant (P301L) tau protein. Nat Genet. 2000 Aug;25(4):402-5. PubMed.
Lewis J, Dickson DW, Lin WL, Chisholm L, Corral A, Jones G, Yen SH, Sahara N, Skipper L, Yager D, Eckman C, Hardy J, Hutton M, McGowan E. Enhanced neurofibrillary degeneration in transgenic mice expressing mutant tau and APP. Science. 2001 Aug 24;293(5534):1487-91. PubMed.
Götz J, Chen F, van Dorpe J, Nitsch RM. Formation of neurofibrillary tangles in P301l tau transgenic mice induced by Abeta 42 fibrils. Science. 2001 Aug 24;293(5534):1491-5. PubMed.

Ordnung, Please—Can Biomarkers Tame a Bewildering Overlap?

03 Jun 2009

Faced with a complicated landscape of mixed disease at all levels of observation, scientists at the 9th International Conference AD/PD last March in Prague made one point abundantly clear. Even as the recognition that neurodegeneration occurs on a spectrum is gaining prominence throughout the field, tangible progress in dealing with spectrum diseases will remain limited until the field comes up with more and better biomarkers of their component proteins. “It is apparent that the next phase of refinements to clinical classification will need to incorporate the use of biological markers of underlying disease process, since clinical presentation alone is an unreliable witness of pathology,” is how Ian McKeith of Newcastle General Hospital in Newcastle upon Tyne, put it in his opening abstract to a workshop in Kassel, Germany, that preceded AD/PD. The same challenge applies to the spectrum of progranulin diseases. Protein-based markers could address the common problem of mis-diagnosis of dementia with Lewy bodies (DLB). In clinical testing, biomarkers could avoid several problems, for example that of trials recruiting patients with different underlying diseases into a single treatment group, or the problem of enrolling patients with simmering preclinical disease into control groups, or of enrolling a person with, e.g., a progranulin-driven dementia into an anti-amyloid drug trial.

What, then, do scientists have in hand? In short, they have candidates in various stages of refinement but no officially validated winners yet. This conference story will summarize some of the imaging markers currently under study for use in diseases of the α-synuclein and progranulin spectrum. The next story will summarize fluid markers (see Part 6 of this series).

First, brain imaging. And first, the bad news. Numerous groups are working on contrast agents and radioligands that would find and label aggregates of α-synuclein and also tau, (see ARF related Eibsee story) but no one appears to have a candidate ready for trial in humans. Michael Pontecorvo of the molecular imaging company AVID Radiopharmaceuticals Inc. is usually a fluent speaker with the polish of a company pitchman. But when asked where things stood on a PET ligand for tau, all he could say was, “We are not close.” For a-synuclein? “Working on it.” How about Aβ oligomers? “Nope… I wish.”

Pontecorvo was more loquacious about dopamine transporter imaging. SPECT scans using ligands for this molecule are already in routine clinical use to diagnose Parkinson disease. In Prague, Pontecorvo presented phase 1 data on an experimental PET ligand for essentially the same purpose. AVID sees advantages because the new agent labels a presymptomatic vesicular monoamine transporter, VMAT2. Its levels decrease with disease but are not up-or downregulated in response to L-Dopa treatment, Pontecorvo said. Called 18F-AV-133 at present, the new ligand enters and leaves the brain rapidly, meaning it could be imaged sooner after the patient receives the injection and would shorten the time the patient has to lie still in the scanner. In a small pilot study, AV-133 distinguished Alzheimer disease (AD) from DLB, Pontecorvo said in Prague. PD and DLB patients both showed a reduction in requisite brain areas, whereas participants even with fairly advanced AD looked like controls. The company also has an amyloid imaging ligand, AV-45 aka Florpiramine, which at present is in a Phase 3 trial and serves as a biomarker in some AD drug trials, though it has no peer-reviewed papers in the scientific literature (see ARF related HAI story). Avid hopes eventually to sell the dopamine transporter ligand and Florpiramine to support differential diagnosis along the spectrum going from AD, DLB, PDD, to PD. “You could scan the same person with both compounds on the same day in three to four hours,” Pontecorvo said.

For his part, David Brooks, who works both at Hammersmith Hospital and for G.E. Healthcare, the commercial developer of Pittsburgh compound B (PIB), reviewed brain imaging approaches for this disease spectrum more broadly. Regarding dopamine transporters (DAT), Brooks cited an older study showing that DAT scans of the striatum distinguish DLB from AD during a person’s life (Walker et al., 2002). Since then, postmortem follow up of people who had undergone DAT scans have shown that whenever the pathologist definitively diagnosed DLB, the person’s DAT scan had been abnormal, whereas when the definitive diagnosis said AD the DAT scan had been normal. A phase 3 multicenter trial further validated this method (see McKeith et al., 2007).

By contrast, on a different method advanced for distinguishing DLB from AD, Brooks noted that his group was unable to reproduce previous data by others. That data had suggested that measuring atrophy in the medial temporal lobe might discriminate (e.g., Burton et al., 2009). This imaging method is more valuable for following disease progression, Brooks said.

Amyloid imaging can be one component of a DLB diagnosis, Brooks said. New Aβ radioligands are joining an increasingly competitive field. The latest is perhaps AstraZeneca’s 11CAZD2184 compound, which Samuel Svensson debuted in Prague (see also Johnson et al., 2009; Svensson comment) These new compounds are just beginning to be tested on a broader scale. The older compound PIB (“older” meaning all of five years) has since 2004 been used at a growing number of independent institutions. It has by now generated a critical mass of data to indicate that, overall, a small majority of patients diagnosed with DLB have brain amyloid loads approaching those of people with AD, Brooks said.

A much smaller percentage of people diagnosed with PDD are PIB-positive. In contrast to DLB, which causes both motor and mental symptoms from the get-go, PDD is a dementia that develops when PD progresses and spreads outward from the nigrostriatal system. PET studies following the fate of dopaminergic and cholinergic neurons show that PDD manifests itself as neuron loss expands from the motor cortex to the parietal and frontal cortex. This causes both a dopaminergic and sweeping cholinergic loss (e.g., Hilker et al., 2005). The former is responsible for increasing disability, the latter for cognitive decline, Brooks concluded. It is clear, however, that “in PDD, the dementia is not caused by amyloid,” he added.

For the purpose of predicting whether a PD patient is likely to develop dementia in the next few years, FDG PET of neuronal activity in cortical areas of the brain appears helpful. Inflammation as imaged with the microglial activation marker 11C-PK11195 also precedes dementia in PD, Brooks said in Prague. Up to 80 percent of people with PD suffer this fate, but typically not before having lived with PD for a decade or more.—Gabrielle Strobel.

This is Part 5 of a nine-part series. See also Part 1, Part 2, Part 3, Part 4, Part 6, Part 7, Part 8, Part 9.

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References

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Paper Citations

Walker Z, Costa DC, Walker RW, Shaw K, Gacinovic S, Stevens T, Livingston G, Ince P, McKeith IG, Katona CL. Differentiation of dementia with Lewy bodies from Alzheimer's disease using a dopaminergic presynaptic ligand. J Neurol Neurosurg Psychiatry. 2002 Aug;73(2):134-40. PubMed.
McKeith I, O'brien J, Walker Z, Tatsch K, Booij J, Darcourt J, Padovani A, Giubbini R, Bonuccelli U, Volterrani D, Holmes C, Kemp P, Tabet N, Meyer I, Reininger C, . Sensitivity and specificity of dopamine transporter imaging with 123I-FP-CIT SPECT in dementia with Lewy bodies: a phase III, multicentre study. Lancet Neurol. 2007 Apr;6(4):305-13. PubMed.
Burton EJ, Barber R, Mukaetova-Ladinska EB, Robson J, Perry RH, Jaros E, Kalaria RN, O'Brien JT. Medial temporal lobe atrophy on MRI differentiates Alzheimer's disease from dementia with Lewy bodies and vascular cognitive impairment: a prospective study with pathological verification of diagnosis. Brain. 2009 Jan;132(Pt 1):195-203. PubMed.
Johnson AE, Jeppsson F, Sandell J, Wensbo D, Neelissen JA, Juréus A, Ström P, Norman H, Farde L, Svensson SP. AZD2184: a radioligand for sensitive detection of beta-amyloid deposits. J Neurochem. 2009 Mar;108(5):1177-86. PubMed.
Hilker R, Thomas AV, Klein JC, Weisenbach S, Kalbe E, Burghaus L, Jacobs AH, Herholz K, Heiss WD. Dementia in Parkinson disease: functional imaging of cholinergic and dopaminergic pathways. Neurology. 2005 Dec 13;65(11):1716-22. PubMed.

External Citations

Still Early Days for α-synuclein Fluid Marker

04 Jun 2009

In Alzheimer disease research, well over a decade of intense research into fluid biomarker candidates has reached a point where a so-called “pathological signature” of amyloid-β and tau proteins is beginning to emerge from the 59-center Alzheimer’s Disease Neuroimaging Study (Shaw et al., 2009). This signature validates on a larger scale a number of earlier studies that had shown essentially the same thing (Fagan et al., 2007; Fagan et al., 2006; Li et al., 2007; Hansson et al., 2006; Welge et al., 2009). Coming, as it did, with ups and downs along the way, this search for a fluid test is guiding researchers who are working to develop similar markers for α-synuclein and progranulin, two major proteins involved in many of the overlapping forms of dementia at issue in earlier parts of this news series. The 9th International Conference AD/PD, held last March in the Czech capital city of Prague, as well as an immediately preceding workshop on dementia with Lewy bodies (DLB) and Parkinson disease dementia (PDD) in Germany, showcased a rapidly growing field of groups who are racing to broaden the field. Here is a selection.

First, α-synuclein (for progranulin, see Part 7). The three investigators who started fluid-based markers on this intraneuronal protein are Michael Schlossmacher, now at the University of Ottawa, Canada, Brit Mollenhauer, now at Paracelsus-Elena-Klinik in Kassel, who worked in Schlossmacher’s former lab at Brigham and Women’s Hospital in Boston, and Omar El-Agnaf, now at United Arab Emirates University in Al Ain, UAE. All three collaborated extensively, first to build ELISA assays and to show that these assays can quantify α-synuclein in normal human cerebrospinal fluid (CSF), then to show that the CSF concentration of this protein normally declines with age and declines even further with Parkinson disease (Tokuda et al., 2006). A first cross-sectional study compared CSF α-synuclein concentration in various patient groups, i.e., AD, DLB, PD, multiple system atrophy (MSA), and Creutzfeldt-Jakob disease (CJD) with controls. These studies found the lowest levels in PD and MSA, whereas AD and controls had similar and higher levels, and DLB lay in between. In CJD, α-synuclein was curiously elevated, perhaps because the rapid cell death in this condition dumps this protein into the CSF so that it serves as a marker of degeneration in this situation, much as tau is viewed in AD (Mollenhauer et al., 2008).

However, the same difficulty that has dogged CSF measurements of Aβ since the beginning (e.g., Seubert et al., 1992) quickly caught up with α-synuclein, too. Its concentration varies greatly from person to person, creating enough overlap that the test in its original form is unable to distinguish which group a given person falls into. While as a group, the values of people with PD always cluster at the bottom, any given person with PD might have a value higher than a control or an AD patient. Moreover, other research groups, using their own, different ELISAs, have been unable so far to replicate Mollenhauer and colleagues’ result, calling into question whether α-synuclein can serve as a robust diagnostic marker to distinguish between overlapping diseases (Ohrfelt et al., 2009; Spies et al. 2009; Noguchi-Shinohara et al., 2009). In Prague, debate centered on the different assays and antibodies different groups are using to measure α-synuclein. Schlossmacher noted that his and collaborators’ ELISA is extensively validated. Other scientists agreed that before a final word can be spoken, more tests in additional patient cohorts, independent replication, a comparison of methods, and exchange of antibodies are needed.

“Right now, many groups are trying to measure CSF α-synuclein and are having a hard time seeing good separation between the groups. We, too, see a very narrow range of values,” said James Galvin of Washington University, St. Louis, Missouri. “But that’s no reason to be discouraged. It may just take more standardization of the steps and the right tools to get it down.”

In Kassel, Mollenhauer presented new data on total CSF α-synuclein measured in a separate cross-sectional cohort of clinically diagnosed patients. Again, PD and MSA lay at the bottom, AD and controls at the top, DLB in between. The overlap remained extensive, though expressing α-synuclein concentration relative to total protein teased the groups apart somewhat. In Prague, Mollenhauer’s poster of a separate series of 41 autopsy-confirmed cases showed that their CSF measurement matched the working diagnosis they had received during life.

On balance, then, the early days of α-synuclein biomarker research have made clear that this protein can be directly measured in the CSF (and peripheral blood; see ARF related news story), and, least with one assay, trends downward from controls to DLB and PD, Schlossmacher said. But besides technical collaboration to streamline protocols, much more scientific work remains to be done, he added. Challenges include understanding where exactly the CSF α-synuclein comes from (the brain, the periphery, the choroid plexus could all contribute), what different species of α-synuclein occur in CSF (truncated, full-length, or modified), and which one of those best indicates disease. To see how these species change in the same person over time, Schlossmacher’s and Mollenhauer’s groups have begun longitudinal studies.

Galvin foresees a future where academic centers interested in earlier-stage clinical trials use a CSF assay for α-synuclein to distinguish preclinical AD from preclinical DLB. An α-synuclein imaging ligand is not on the horizon (see Part 5 of this series), but amyloid imaging is available and it shows a large fraction of non-demented elderly people who have brain amyloid and may turn out to be presymptomatic for either AD or DLB. Most DLB cases share Aβ and α-synuclein pathology; hence, an α-synuclein fluid assay could conceivably flag amyloid-positive people who are at high risk for future DLB, much like combining amyloid imaging with CSF Aβ/tau measurement is predicting who will develop AD symptoms. Other groups are drilling deeper with Aβ biochemistry, measuring some of its truncated and oxidized forms to distinguish between AD and DLB (Bibl et al., 2006; Bibl et al., 2007).

“In our studies, we already have a number of people who are PIB positive and are not demented, but when you look at them with some of the biomarkers we are developing, some of these people are clustering with the DLB group. The idea is to be able to diagnose preclinical DLB,” Galvin said. The Kassel meeting ended with the designation of a working group to hammer out a research path toward that goal, Mollenhauer wrote to ARF.

For his part, El-Agnaf has focused on measuring oligomers of α-synuclein, initially in plasma (El-Agnaf et al., 2006) and more recently in brain extracts. In Prague, he showed the results of a study looking for such oligomers in lysates prepared from postmortem brains of people who had suffered from DLB. As measured by a sandwich ELISA El-Agnaf developed with a commercial antibody that recognizes α-synuclein aggregates but not monomers, these brains contained far higher concentrations of α-synuclein oligomers than control or AD brains. The data showed less overlap between the groups, but no clear separation, either (Paleologou et al., 2009). Since then, the researchers used their ELISA on CSF samples and again found high levels. A final cohort of 60 samples from people with PD and controls displayed, again, a group difference but also a spread of the individual data points and overlap between the groups. Calculating the ratio of oligomeric α-synuclein to total α-synuclein improved the separation, El-Agnaf noted. “This is the first time we have been able to detect soluble oligomers from CSF in humans,” El-Agnaf said in Prague, and here, too, the work of replication and broadening the effort is only just beginning.

Meanwhile, research underpinning the rationale for going after oligomeric α-synuclein in body fluids is advancing in parallel. Here, too, Prague offered some news. For example, in the last talk of the AD/PD conference, Kostas Vekrellis of the Biomedical Research Foundation Academy of Athens, Greece, reported that secreted α-synuclein oligomers are up to no good. Even though α-synuclein is primarily a cytosolic protein, scientists know that cultured cells can release it. Cells also can take up external α-synuclein, usually at their peril as they tend to die soon after, Vekrellis said.

Vekrellis investigated this apparent toxicity with lines of human neuroblastoma cells that can be induced to express wild-type α-synuclein. Soluble monomeric and oligomeric α-synuclein showed up in the conditioned medium from these cells. Using liquid chromatography-mass spectrometry proteomics and electron microscopy, Vekrellis and colleagues showed that the cells actively export α-synuclein via an exosome pathway that itself depends on intracellular calcium. These cells sustained no harm from the α-synuclein. But their conditioned medium, when squirted onto primary rat cortical neurons, killed those cells. A high-molecular-weight fraction of α-synuclein species proved toxic. Medium depleted of α-synuclein, or medium subjected to oligomer-busting compounds such as scylloinositol did not, pointing to oligomers as the active component. This new data invoke parallels with amyloid-β oligomers, which have been shown to impair synaptic activity and to damage cells, and whose sensitivity to scylloinositol has led to a Phase 2 trial. For more on oligomers in mixed disease, see Part 4 of this series.—Gabrielle Strobel.

This is Part 6 of a nine-part series. See also Part 1, Part 2, Part 3, Part 4, Part 5, Part 7, Part 8, Part 9.

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References

News Citations

Paper Citations

Shaw LM, Vanderstichele H, Knapik-Czajka M, Clark CM, Aisen PS, Petersen RC, Blennow K, Soares H, Simon A, Lewczuk P, Dean R, Siemers E, Potter W, Lee VM, Trojanowski JQ, Alzheimer's Disease Neuroimaging Initiative. Cerebrospinal fluid biomarker signature in Alzheimer's disease neuroimaging initiative subjects. Ann Neurol. 2009 Apr;65(4):403-13. PubMed.
Fagan AM, Roe CM, Xiong C, Mintun MA, Morris JC, Holtzman DM. Cerebrospinal fluid tau/beta-amyloid(42) ratio as a prediction of cognitive decline in nondemented older adults. Arch Neurol. 2007 Mar;64(3):343-9. Epub 2007 Jan 8 PubMed.
Fagan AM, Mintun MA, Mach RH, Lee SY, Dence CS, Shah AR, Larossa GN, Spinner ML, Klunk WE, Mathis CA, Dekosky ST, Morris JC, Holtzman DM. Inverse relation between in vivo amyloid imaging load and cerebrospinal fluid Abeta42 in humans. Ann Neurol. 2006 Mar;59(3):512-9. PubMed.
Li G, Sokal I, Quinn JF, Leverenz JB, Brodey M, Schellenberg GD, Kaye JA, Raskind MA, Zhang J, Peskind ER, Montine TJ. CSF tau/Abeta42 ratio for increased risk of mild cognitive impairment: a follow-up study. Neurology. 2007 Aug 14;69(7):631-9. PubMed.
Hansson O, Zetterberg H, Buchhave P, Londos E, Blennow K, Minthon L. Association between CSF biomarkers and incipient Alzheimer's disease in patients with mild cognitive impairment: a follow-up study. Lancet Neurol. 2006 Mar;5(3):228-34. PubMed.
Welge V, Fiege O, Lewczuk P, Mollenhauer B, Esselmann H, Klafki HW, Wolf S, Trenkwalder C, Otto M, Kornhuber J, Wiltfang J, Bibl M. Combined CSF tau, p-tau181 and amyloid-beta 38/40/42 for diagnosing Alzheimer's disease. J Neural Transm. 2009 Feb;116(2):203-12. Epub 2009 Jan 14 PubMed.
Tokuda T, Salem SA, Allsop D, Mizuno T, Nakagawa M, Qureshi MM, Locascio JJ, Schlossmacher MG, El-Agnaf OM. Decreased alpha-synuclein in cerebrospinal fluid of aged individuals and subjects with Parkinson's disease. Biochem Biophys Res Commun. 2006 Oct 13;349(1):162-6. PubMed.
Mollenhauer B, Cullen V, Kahn I, Krastins B, Outeiro TF, Pepivani I, Ng J, Schulz-Schaeffer W, Kretzschmar HA, McLean PJ, Trenkwalder C, Sarracino DA, Vonsattel JP, Locascio JJ, El-Agnaf OM, Schlossmacher MG. Direct quantification of CSF alpha-synuclein by ELISA and first cross-sectional study in patients with neurodegeneration. Exp Neurol. 2008 Oct;213(2):315-25. PubMed.
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Ohrfelt A, Grognet P, Andreasen N, Wallin A, Vanmechelen E, Blennow K, Zetterberg H. Cerebrospinal fluid alpha-synuclein in neurodegenerative disorders-a marker of synapse loss?. Neurosci Lett. 2009 Feb 6;450(3):332-5. PubMed.
Spies PE, Melis RJ, Sjögren MJ, Rikkert MG, Verbeek MM. Cerebrospinal fluid alpha-synuclein does not discriminate between dementia disorders. J Alzheimers Dis. 2009;16(2):363-9. PubMed.
Noguchi-Shinohara M, Tokuda T, Yoshita M, Kasai T, Ono K, Nakagawa M, El-Agnaf OM, Yamada M. CSF alpha-synuclein levels in dementia with Lewy bodies and Alzheimer's disease. Brain Res. 2009 Jan 28;1251:1-6. PubMed.
Bibl M, Mollenhauer B, Esselmann H, Lewczuk P, Klafki HW, Sparbier K, Smirnov A, Cepek L, Trenkwalder C, Rüther E, Kornhuber J, Otto M, Wiltfang J. CSF amyloid-beta-peptides in Alzheimer's disease, dementia with Lewy bodies and Parkinson's disease dementia. Brain. 2006 May;129(Pt 5):1177-87. PubMed.
Bibl M, Mollenhauer B, Lewczuk P, Esselmann H, Wolf S, Trenkwalder C, Otto M, Stiens G, Rüther E, Kornhuber J, Wiltfang J. Validation of amyloid-beta peptides in CSF diagnosis of neurodegenerative dementias. Mol Psychiatry. 2007 Jul;12(7):671-80. PubMed.
El-Agnaf OM, Salem SA, Paleologou KE, Curran MD, Gibson MJ, Court JA, Schlossmacher MG, Allsop D. Detection of oligomeric forms of alpha-synuclein protein in human plasma as a potential biomarker for Parkinson's disease. FASEB J. 2006 Mar;20(3):419-25. PubMed.
Paleologou KE, Kragh CL, Mann DM, Salem SA, Al-Shami R, Allsop D, Hassan AH, Jensen PH, El-Agnaf OM. Detection of elevated levels of soluble alpha-synuclein oligomers in post-mortem brain extracts from patients with dementia with Lewy bodies. Brain. 2009 Apr;132(Pt 4):1093-101. PubMed.

External Citations

Meet Progranulin, The Biomarker—A Simpler Story?

05 Jun 2009

Scientists wrestling the complexities of α-synuclein fluid biochemistry might be forgiven for looking with some envy to a different protein of the neurodegenerative disease spectrum. At the 9th International Conference AD/PD, held last March in Prague, the field learned that crafting such a test for progranulin might actually be comparatively easy—incredible as that sounds in the field of neurodegeneration where generally speaking nothing is easy. Progranulin is the protein behind a sizable fraction of frontotemporal dementia and probably also a small, still-unknown fraction of cases diagnosed clinically as early onset Alzheimer’s or related disorders. In Prague, three independent groups presented results of their fledgling ELISA tests—one in serum, one in plasma, and one in cerebrospinal fluid. Incredible as it may seem to a biomarker field plagued by inconsistent findings on blood tests for other proteins such as Aβ, all three groups reported the same overall result: it works just fine, thank you.

Progranulin surfaced independently in the laboratories of Christine van Broeckhoven at the VIB-University of Antwerp, Belgium, and of Michael Hutton, then at the Mayo Clinic Jacksonville, Florida, as the gene for the tau-negative form of frontotemporal dementia 17 (FTLD-U). This highly familial disease frequently strikes people younger than 65 (see ARF related news story). Since then, 66 pathogenic mutations in progranulin have turned up (see mutation database). Importantly, progranulin causes neurodegeneration by a different mechanism than Aβ, tau, or α-synuclein (see Part 1 of this series). Whereas these latter proteins are thought to become toxic as their concentration rises (i.e., the more protein, the earlier one gets sick), progranulin leads to neurodegeneration when there is not enough of it. With Aβ, tau, and α-synuclein, mutations that drastically increase expression cause familial early onset disease, whereas risk alleles influence sporadic disease. In contrast, the general theme emerging from progranulin genetics is that null mutations that slash protein levels in half cause familial FTLD-U, while milder missense mutations that cause a partial loss of function have a susceptibility role in Alzheimer’s, amyotrophic lateral sclerosis, and perhaps Parkinson disease, van Broeckhoven said in her talk in Prague.

Progranulin’s different mechanism should translate into differences in diagnosis and treatment. The gene itself is plenty complicated, and the six different granulin proteins resulting from it have physiological functions throughout the body. But diagnosis might be straightforward. “We thought progranulin protein levels should be decreased in the blood of people with mutations that cause loss of function,” Kristel Sleegers in van Broeckhoven’s group said in her talk. Sleegers started with an ELISA against full-length progranulin developed originally by Philip van Damme (van Damme et al., 2008). She put it to work on blood samples from a large Belgian founder family whose 43 patients showcase the dramatic clinical heterogeneity of progranulin mutations. Their clinical diagnoses range from FTD, AD, PD, primary progressive aphasia (PPA), and progressive nonfluent aphasia (PNFA)—all from having inherited the same mutation. Pathologically, this family runs the gamut, too, with Lewy bodies, ubiquitin-positive FTLD-U inclusions, amyloid pathology, and of course TDP-43 (Brouwers et al., 2007).

From this family, Sleegers had serum of six patients, eight younger still-unaffected mutation carriers, and nine non-carriers. The ELISA distinguished carriers and non-carriers unequivocally, Sleegers showed. The groups were completely separate and apart by a large distance. Interestingly, the non-symptomatic carriers had the same progranulin levels as their affected relatives, suggesting the ELISA may be able to detect preclinical disease and presymptomatic mutation carriers. Genetic testing can do this, too, but it is more complicated to interpret, as scientists need to know whether a change in the gene sequence is pathogenic or a harmless variation, and genetic deletions require further analysis. Besides capturing all types of progranulin mutation, a blood-based ELISA could also be cheaper than genetic testing.

In her talk, Rosa Rademakers of the Mayo Clinic Jacksonville, Florida, reported the same results in a different, larger group of patients. Rademakers is a neurogeneticist also formerly of van Broeckhoven’s group; she received a Young Investigator Award at the conference (see ARF related news story). Her team optimized a commercial ELISA for human progranulin and tested plasma of 219 patients clinically diagnosed with FTD. In this study, too, all patients carrying a loss-of-function progranulin mutation had only about one-third as much progranulin in their blood as did patients without a progranulin mutation. The ELISA predicted with 100 percent certainty that everyone with less than 112 nanogram/milliliter (ng/ml) of the growth factor carried a progranulin mutation. This is nearly identical to the cutoff suggested in an earlier Italian study led by Giuliano Binetti at the Centro San Giovanni di Dio-Fatebenefratelli in Brescia, which tested plasma and CSF ELISAs in a group of FTLD patients (Ghidoni et al., 2008).

Working in parallel, both groups next studied whether their ELISAs could tease apart some of the multiple disease processes underlying a clinically defined disease, in other words, serve as a new tool to better define the spectrum of neurodegeneration. For example, Sleegers reported that progranulin was low in the serum of a person who had been diagnosed with AD but later proved to have a loss-of-function progranulin mutation. Conversely, Rademakers showed that the plasma test revealed abnormally low progranulin in one of 72 people clinically diagnosed with AD and that this man, upon sequencing, proved to have a new loss-of-function progranulin mutation. Likewise, a French man with clinical Parkinson disease and a progranulin mutation also had low plasma progranulin. “Regardless of how a person presents clinically, the ELISA detects a progranulin null mutation,” Sleegers said.

Lastly, both studies explored whether their ELISAs were able to pick up more subtle genetic flaws in progranulin. For example, missense mutations cause less than haploinsufficiency, which results from a mutation that aborts protein production entirely. Researchers are exploring different kinds of missense mutations in this gene. Some hasten the degradation of the protein, others reduce its secretion, and in-silico modeling points to misfolding at the protein’s internal disulfide bridges as a possible cause for these cellular problems with the protein (Shankaran et al, 2007; van der Zee et al., 2007). In Prague, Sleegers closed her talk with data showing that both in people with clinical FDT and AD, missense mutations that such research had predicted to be pathogenic came with reduced serum progranulin levels in their carriers, though the drop was less precipitous than with a null mutation. Missense mutations predicted to be harmless corresponded to normal levels of serum progranulin. Both Sleegers’ and Rademaker’s studies appeared recently online (Sleegers et al., 2009; Finch et al., 2009).

Last but not least, also in Prague, German researchers led by Anja Capell and Christian Haass at Ludwig-Maximilian University in Munich presented ongoing work on a third ELISA to quantify progranulin in the CSF. Compared to serum and plasma, where progranulin levels ranged in the low hundreds of ng/ml for controls and 50 to 90 ng/ml in null mutation carriers, CSF levels are much lower, around 5-7 ng/ml in controls. Previous work has reported this same range in controls and about 2 ng/ml in mutation carriers (van Damme et al., 2008). In clinical practice, it is not clear if a spinal tap will be necessary eventually, because blood-based tests appear to work well so far, Rademakers wrote by e-mail.

All told, these studies suggest that blood tests could reveal an underlying progranulin-driven disease process regardless of how it manifests clinically. It is simpler than genetics because it picks up the loss of the protein no matter which of a myriad of different genetic changes might be to blame. Such a blood test could show whose early onset dementia is due to this particular protein, and predict future neurodegeneration in people who are still cognitively healthy but at risk because of their family history. Viewed broadly beyond FTD, progranulin tests could help explain a slice of the neurodegenerative disease spectrum.—Gabrielle Strobel.

This is Part 7 of a nine-part series. See also Part 1, Part 2, Part 3, Part 4, Part 5, Part 6, Part 8, Part 9.

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References

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Brouwers N, Nuytemans K, van der Zee J, Gijselinck I, Engelborghs S, Theuns J, Kumar-Singh S, Pickut BA, Pals P, Dermaut B, Bogaerts V, De Pooter T, Serneels S, Van den Broeck M, Cuijt I, Mattheijssens M, Peeters K, Sciot R, Martin JJ, Cras P, Santens P, Vandenberghe R, De Deyn PP, Cruts M, Van Broeckhoven C, Sleegers K. Alzheimer and Parkinson diagnoses in progranulin null mutation carriers in an extended founder family. Arch Neurol. 2007 Oct;64(10):1436-46. PubMed.
Ghidoni R, Benussi L, Glionna M, Franzoni M, Binetti G. Low plasma progranulin levels predict progranulin mutations in frontotemporal lobar degeneration. Neurology. 2008 Oct 14;71(16):1235-9. PubMed.
Shankaran SS, Capell A, Hruscha AT, Fellerer K, Neumann M, Schmid B, Haass C. Missense mutations in the progranulin gene linked to frontotemporal lobar degeneration with ubiquitin-immunoreactive inclusions reduce progranulin production and secretion. J Biol Chem. 2008 Jan 18;283(3):1744-53. Epub 2007 Nov 5 PubMed.
van der Zee J, Le Ber I, Maurer-Stroh S, Engelborghs S, Gijselinck I, Camuzat A, Brouwers N, Vandenberghe R, Sleegers K, Hannequin D, Dermaut B, Schymkowitz J, Campion D, Santens P, Martin JJ, Lacomblez L, De Pooter T, Peeters K, Mattheijssens M, Vercelletto M, Van den Broeck M, Cruts M, De Deyn PP, Rousseau F, Brice A, Van Broeckhoven C. Mutations other than null mutations producing a pathogenic loss of progranulin in frontotemporal dementia. Hum Mutat. 2007 Apr;28(4):416. PubMed.
Sleegers K, Brouwers N, Van Damme P, Engelborghs S, Gijselinck I, van der Zee J, Peeters K, Mattheijssens M, Cruts M, Vandenberghe R, De Deyn PP, Robberecht W, Van Broeckhoven C. Serum biomarker for progranulin-associated frontotemporal lobar degeneration. Ann Neurol. 2009 May;65(5):603-9. PubMed.

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Reshuffle Parkinson’s Genetics to Lay Out Its Pathways?

09 Jun 2009

This penultimate article in the Alzforum series on the spectrum of overlapping neurodegenerative diseases reports the call of a founding neurogeneticist, who rocks the boat from time to time, to reorganize the genetics of Parkinson’s and related diseases. In a series of provocative talks, including one at a workshop on Dementia with Lewy Bodies and Parkinson’s Disease Dementia held last March in the German city of Kassel, and again in a plenary thereafter at the 9th International conference AD/PD 2009 in Prague, John Hardy of University College London, UK, called on his audiences to cast a critical eye over the current list of proposed Parkinson’s genes and engage in some spring cleaning. Out with some genes, in with others, and the field could benefit by seeing—voila—a common metabolic pathway for Lewy body disorders, Hardy claimed.

In essence, the idea is that if three genes currently regarded as PD genes were instead set aside as genes for a mitochondrial path to neuronal death in the substantia nigra, and if other genes known to cause clinically different diseases that nonetheless come with Lewy body pathology were included, then the field might make faster progress in understanding molecular mechanisms of Lewy body diseases. That’s because scientists would have a list of genes that all impinge in some way on the metabolism of the brain glycolipid ceramide. Two papers out this month in Archives of Neurology on one such gene—glucocerebrosidase (GBA)—support this claim by placing GBA front and center as the strongest risk factor to date for sporadic PD and DLB (Mitsui et al., 2009; Clark et al., 2009; Leverenz et al., 2009; covered in Part 9 of this series).

With such a reorganization of the PARK loci, Hardy said, scientists would avoid being misled by a bewildering variety of clinical parkinsonian phenotypes. Rather than following clinical descriptions, gene sleuths could instead grab hold of the pathology as a starting point to find the underlying gene mutations and risk variants that drive pathogenesis. In a review article published this month, Hardy and colleagues recall that basing genetics on pathology has served Alzheimer disease research well (Hardy et al., 2009). Starting with Alois Alzheimer, AD was defined from the get-go by its definitive pathology of plaques and tangles. This helped geneticists find the autosomal-dominant genes, and the molecular biology of AD took off as a field when those three genes turned out to fall into the very pathway of APP metabolism that George Glenner had anticipated with his biochemical isolation of amyloid from brain. Other clinical dementias—e.g., vascular, frontotemporal—were declared different and thus did not distract the geneticists. “Just think if we had gone for genes for dementia, not for Alzheimer disease. We would be in a terrible mess,” Hardy wrote to ARF.

Parkinson disease has a different history. First, unlike Alzheimer’s 1906 paper, James Parkinson’s eponymous description, in 1817, of the shaking palsy was clinical. Second, the breakthrough of L-Dopa therapy in the 1960s rightly reinforced the importance of recognizing the clinical features because patients responded so well to this symptomatic drug, the scientists write in their review. Later it became clear that most people with PD have Lewy bodies. But so do people who suffer from other clinical diseases, and the pathology in PD generally took a back seat to the clinical perspective. While clinical classification is fine for treatment, however, it can lead molecular pathogenesis research into a thicket and delay the delineation of the multiple protein-driven pathways that give rise, either alone or in combination, to a person’s individual combination of clinical symptoms, Hardy claims.

Such a reassessment is timely now. It would cap a decade during which genetics has risen to prominence in PD research. Before that, Parkinson’s was widely considered a sporadic disease, but the initial discovery in 1997 of an a-synuclein mutation in a Greek/Italian family with early onset PD triggered a period of genetic inquiry that currently amounts to 457 genes being tracked in the PDGene database.

Here’s a current selection of the best-known PARK loci Hardy showed in Prague:

What does he find wrong with this picture? The right column shows that three well-known genes generally cause no Lewy body pathology or at least have not been reported as such yet. Parkin, Pink-1, and DJ-1 are inherited in an autosomal-recessive mode, and generally come as loss-of-function alleles. To the geneticist, proof of pathogenicity for some of those, particularly missense alleles, can be difficult to establish. Yet the primary reason Hardy suggests for grouping these three separately is that they appear to act in the same underlying pathway that is separate from the pathway of α-synuclein, LRRK2, and the new slugger in PD/DLB genetics, GBA (see part 9). Pink-1 acts upstream of parkin in the same mitochondrial pathway; DJ-1, while not directly placed in the same pathway, is an essential part of the oxidative stress response in mitochondria, too. Clinically, mutations in these three genes tend to give rise to very early onset syndromes of juvenile parkinsonism starting, in rare cases, even in childhood. In essence, Hardy suggests this: these genes are important, but they cause a mitochondrial path to parkinsonian symptoms that is probably separate from a main PD/DLB pathway that gives rise to the predominant pathology of intracellular α-synuclein aggregates. Hence, Hardy moves them to the left in his suggested pathway diagram below:

View larger image.

With the 3 green mitochondrial genes on the left, what’s happening on the right? Hardy suggested that the protein aggregation that leads to Lewy bodies and Lewy neurites (the main types of α-synuclein pathology visible to microcopy) encompasses a set of genes that are not typically seen as belonging to the PD spectrum, but deserve consideration. First, three smaller ones, then the big one. The little-known PLA2G6 gene causes an adult-onset parkinsonian movement disorder with cognitive and psychiatric symptoms, and the PANK2 gene causes a severe movement disorder with mental deterioration called Hallervorden-Spatz. Importantly, both of these rare diseases have Lewy pathology (Wakabayashi et al., 1999; Zhou et al., 2001; Paisan-Ruiz et al., 2008). Their proteins both also happen to map to different arms of lysosomal ceramide metabolism. When they function normally, they reduce levels of this lipid (see diagram below by Jose Bras in Hardy’s group).

View larger image.

Then there is the better-known Niemann-Pick Type C (NPC) gene. Recessive mutations cause the childhood-onset lysosomal storage disease known for its progressive neurodegeneration, though its phenotype varies widely. This disease might fit in because it also features Lewy bodies, and the NPC mutant from patients is known to reduce the activity of acid sphingomyelinase, an enzyme in this pathway, Hardy writes in the review. The physiological function of α-synuclein, the founding member of Lewy body disease genetics and the protein at the heart of its pathology, is still largely a mystery. But besides its synaptic localization, a biochemical role in brain lipid metabolism is likely, Hardy said (e.g., Golovko et al., 2006).

The big new anchor for this scenario, however, is GBA, the gene for the enzyme glucocerebrosidase. Five new papers published over the last 2 months alone strengthen its central role in the genetics of Lewy body diseases. Its story concludes this news series.—Gabrielle Strobel.

This is Part 8 of a nine-part series. See also Part 1, Part 2, Part 3, Part 4, Part 5, Part 6, Part 7, Part 9.

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References

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Clark LN, Kartsaklis LA, Wolf Gilbert R, Dorado B, Ross BM, Kisselev S, Verbitsky M, Mejia-Santana H, Cote LJ, Andrews H, Vonsattel JP, Fahn S, Mayeux R, Honig LS, Marder K. Association of glucocerebrosidase mutations with dementia with lewy bodies. Arch Neurol. 2009 May;66(5):578-83. PubMed.
Leverenz JB, Lopez OL, Dekosky ST. The expanding role of genetics in the lewy body diseases: the glucocerebrosidase gene. Arch Neurol. 2009 May;66(5):555-6. PubMed.
Hardy J, Lewis P, Revesz T, Lees A, Paisan-Ruiz C. The genetics of Parkinson's syndromes: a critical review. Curr Opin Genet Dev. 2009 Jun;19(3):254-65. PubMed.
Wakabayashi K, Yoshimoto M, Fukushima T, Koide R, Horikawa Y, Morita T, Takahashi H. Widespread occurrence of alpha-synuclein/NACP-immunoreactive neuronal inclusions in juvenile and adult-onset Hallervorden-Spatz disease with Lewy bodies. Neuropathol Appl Neurobiol. 1999 Oct;25(5):363-8. PubMed.
Zhou B, Westaway SK, Levinson B, Johnson MA, Gitschier J, Hayflick SJ. A novel pantothenate kinase gene (PANK2) is defective in Hallervorden-Spatz syndrome. Nat Genet. 2001 Aug;28(4):345-9. PubMed.
Paisan-Ruiz C, Bhatia KP, Li A, Hernandez D, Davis M, Wood NW, Hardy J, Houlden H, Singleton A, Schneider SA. Characterization of PLA2G6 as a locus for dystonia-parkinsonism. Ann Neurol. 2009 Jan;65(1):19-23. PubMed.
Golovko MY, Rosenberger TA, Faergeman NJ, Feddersen S, Cole NB, Pribill I, Berger J, Nussbaum RL, Murphy EJ. Acyl-CoA synthetase activity links wild-type but not mutant alpha-synuclein to brain arachidonate metabolism. Biochemistry. 2006 Jun 6;45(22):6956-66. PubMed.

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More Than Gaucher’s—GBA Throws Its Weight Around Lewy Body Disease

09 Jun 2009

This news story on the glucocerebrosidase (GBA) gene closes the Alzforum series on emerging concepts in the neurodegenerative disease spectrum. This latest genetics discovery may help scientists distinguish cases on the Lewy body end of the spectrum (i.e., toward Parkinson disease and some dementia with Lewy bodies) from the plaque and tangle end of the spectrum (i.e., Alzheimer disease). This year alone has seen five original research papers and several reviews on the growing realization that heterozygous mutations and pathogenic alleles of this enzyme are the most prevalent risk factor known to date for PD and other Lewy body diseases. “This is a big story,” said John Hardy, who argued at recent conferences that the GBA gene deserves a central place in the genetic lineup of Lewy body disease genes (see Part 8). These days, GBA ranks first on the Top PDGene Results, though these rankings change constantly with additional findings. Even since that database was last updated, a torrent of new data on larger patient groups and a greater number of rare disease variants appeared online this month. It further solidifies the position of the Gaucher disease gene as a major risk gene for sporadic and even familial clustering of these diseases. “We can have confidence that GBA has an important role in the pathogenesis of Lewy body disorders,” wrote James Leverenz of University of Washington, Seattle, and colleagues in an editorial accompanying the latest two papers in Archives of Neurology (Leverenz et al., 2009).

Until recently, GBA was known primarily for causing Gaucher disease. This is an autosomal recessive, lysosomal storage disease of glucocerebrosidase deficiency. It can cause acute liver damage even early in life, typically in homozygous mutation carriers, when the substrate of the mutated, sluggish enzyme—the lipid glucocerebroside—accumulates in cells. At some 10,000 estimated cases worldwide, Gaucher’s is an orphan disease. The gradual expansion of GBA’s relevance to a much larger group of people historically began with case reports of parkinsonism in Ashkenazi Jewish patients with Gaucher disease (Neudorfer et al., 1996; Machaczka et al., 1999). These reports initially drew little attention among PD epidemiologists and geneticists, said Hardy. More widely noted, at least in the U.S., were papers by Ellen Sidransky at the NIH in Bethesda, Maryland, who pursued her clinical observation that fathers and uncles of her patients with Gaucher’s tended to show parkinsonian symptoms. Sidransky’s group conducted a series of small studies looking at neuropathology and GBA mutations in family members (Tayebi et al., 2001; Wong et al., 2004; Tayebi et al., 2003; Lwin et al., 2004).

Together, the Israeli and U.S. work inspired groups worldwide to look for GBA mutations in PD patient series of non-Ashkenazi origin. These studies were generally small and only assessed specific GBA SNPs known from Gaucher disease, not the entire sequence of the gene, but even so, many of them were positive. Overall, this existing work led to a sense that the acute liver problems of Gaucher’s develop when a person lacks at least 80 percent of GBA activity, whereas milder, or the more common heterozygous mutations that eliminate about half of the body’s GBA activity enable a healthy childhood but can cause later-onset Lewy body diseases such as PD or dementia with Lewy bodies, Hardy and colleagues write in a review out this month (Hardy et al., 2009).

In the past three months, the story suddenly bulked up when data on larger patient series pouring in. In Prague, Laura Parkkinen of University College London, UK, presented that group’s latest results on 790 patients with PD and 257 controls. Four percent of the patients had one of 14 different GBA mutations, adding up to a total odds ratio of 3.7. All GBA carriers who had consented to autopsy showed extensive Lewy body pathology in their brain. Clinically, about half had typical PD; the other half also had the hallucinations and cognitive decline that marks dementia with Lewy bodies (DLB, see part 3 of this series). “GBA mutations are the most common genetic risk factor for developing sporadic PD or DLB in this large British population,” Parkkinen said. This data appeared last March (Neumann et al., 2009), as did the results of a separate series of 172 Greek Parkinson’s patients in whom GBA also proved the most commonly mutated gene, amounting to a similar odds ratio of 4.2 (Kalinderi et al., 2009).

Still-larger datasets rolled in this month from Japan and New York. Led by Shoji Tsuji at University of Tokyo Graduate School of Medicine, first author Jun Mitsui and collaborators at other Japanese institutions reported that their re-sequencing effort of the entire GBA gene in 534 PD patients and 544 controls discovered 11 pathogenic variants that together occurred in a total of 10 percent of patients but in almost no controls, leading to a whopping odds ratio of 28 (Mitsui et al., 2009). The British, the Greek, and the Japanese studies, as had some previous ones, all found that GBA mutations showed up particularly in early onset patients. The large patient group in the Japanese sample included 34 families with clusters of PD cases; of those families, eight had heterozygous GBA variants that showed up in all affected relatives, making GBA a gene not only for sporadic PD, but also for some forms of autosomal-recessive familial PD.

These data imply that the field will have to abandon the comparatively simple idea that common diseases like PD are caused by common gene variants. Instead geneticists are coming to grips with the more complex notion that common diseases are caused by many different variants, many of which will be rare. “We should emphasize a paradigm shift from the common disease—common variants hypothesis to the common disease—multiple rare variants hypothesis in our search for disease susceptibility genes in sporadic PD, which may be applicable to studies of other diseases,” Mitsui and colleagues wrote. In practice, this paradigm shift amounts to a tall order, as finding these multiple rare variants requires extensive sequencing of the entire gene in cases as well as in many controls. For GBA, this is difficult because the existence of an adjacent pseudogene makes this whole genomic region challenging to dissect.

For its part, the New York City study, led by Karen Marder at Columbia University, focused on patients with dementia with Lewy bodies (DLB). Of 95 people who had pathologically confirmed Lewy body disease, fully 28 percent had GBA mutations. Relatively fewer, that is, 10 percent, of people who had AD pathology also had GBA mutations, as did 3 percent of controls without either AD or Lewy body pathology. These authors report that in their patient sample, GBA mutations tended to lead to extensive α-synuclein pathology in the cortex. They suggest that in this way, GBA might serve as a diagnostic marker during life, indicating that mutation carriers likely have “purer” Lewy body pathology and that their dementia results primarily from that, not from the amyloid and tau pathology that marks AD (Clark et al., 2009).

How GBA variants cause PD and DLB is unknown at this point, because the molecular work studying the variants remains to be done. However, in these early days, most scientists interviewed for this article leaned toward a loss-of function mechanism. Some scientists noted that research into whether glucocerebrosidase variants might impair protein degradation in lysosomes might lead to new insights about α-synuclein processing and aggregation. Others raised the notion that GBA enzyme activity could provide a basis for developing fluid biomarkers (e.g., Balducci et al., 2007), similarly to the way they are coming along for progranulin, a growth factor whose own loss of function is genetically linked to many cases of frontotemporal dementia (see Part 7). That is in the future; but even now, scientists agree that recent genetics news have noticeably shifted the PD and DLB landscape toward lysosomal ceramide metabolism as a promising area of research (see series Part 8; also Depaolo et al., 2009).—Gabrielle Strobel.

This is Part 9 of a nine-part series. See also Part 1, Part 2, Part 3, Part 4, Part 5, Part 6, Part 7, Part 8.

References

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Leverenz JB, Lopez OL, Dekosky ST. The expanding role of genetics in the lewy body diseases: the glucocerebrosidase gene. Arch Neurol. 2009 May;66(5):555-6. PubMed.
Neudorfer O, Giladi N, Elstein D, Abrahamov A, Turezkite T, Aghai E, Reches A, Bembi B, Zimran A. Occurrence of Parkinson's syndrome in type I Gaucher disease. QJM. 1996 Sep;89(9):691-4. PubMed.
Machaczka M, Rucinska M, Skotnicki AB, Jurczak W. Parkinson's syndrome preceding clinical manifestation of Gaucher's disease. Am J Hematol. 1999 Jul;61(3):216-7. PubMed.
Tayebi N, Callahan M, Madike V, Stubblefield BK, Orvisky E, Krasnewich D, Fillano JJ, Sidransky E. Gaucher disease and parkinsonism: a phenotypic and genotypic characterization. Mol Genet Metab. 2001 Aug;73(4):313-21. PubMed.
Wong K, Sidransky E, Verma A, Mixon T, Sandberg GD, Wakefield LK, Morrison A, Lwin A, Colegial C, Allman JM, Schiffmann R. Neuropathology provides clues to the pathophysiology of Gaucher disease. Mol Genet Metab. 2004 Jul;82(3):192-207. PubMed.
Tayebi N, Walker J, Stubblefield B, Orvisky E, LaMarca ME, Wong K, Rosenbaum H, Schiffmann R, Bembi B, Sidransky E. Gaucher disease with parkinsonian manifestations: does glucocerebrosidase deficiency contribute to a vulnerability to parkinsonism?. Mol Genet Metab. 2003 Jun;79(2):104-9. PubMed.
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Hardy J, Lewis P, Revesz T, Lees A, Paisan-Ruiz C. The genetics of Parkinson's syndromes: a critical review. Curr Opin Genet Dev. 2009 Jun;19(3):254-65. PubMed.
Neumann J, Bras J, Deas E, O'Sullivan SS, Parkkinen L, Lachmann RH, Li A, Holton J, Guerreiro R, Paudel R, Segarane B, Singleton A, Lees A, Hardy J, Houlden H, Revesz T, Wood NW. Glucocerebrosidase mutations in clinical and pathologically proven Parkinson's disease. Brain. 2009 Jul;132(Pt 7):1783-94. PubMed.
Kalinderi K, Bostantjopoulou S, Paisan-Ruiz C, Katsarou Z, Hardy J, Fidani L. Complete screening for glucocerebrosidase mutations in Parkinson disease patients from Greece. Neurosci Lett. 2009 Mar 13;452(2):87-9. PubMed.
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Prague: AD/PD Convenes Record Number of Youngsters and Leaders

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Prague: What Say You, Alois—Should It Be “Alzheimer-Fischer” Disease?

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Prague: N-terminal APP Story Buzzes in the Hallways

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Prague: Tau-Laden Neurons in Zebrafish Glow, Perish on Candid Camera

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Prague: Aβ Rehabilitated as an Antimicrobial Protein?

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Prague: Piecing Together Pathology with PyroGlu

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Spectrum of Neurodegeneration Comes to the Fore

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Et tu, Brute? Parkinson’s GWAS Fingers Tau Next to α-Synuclein

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Neither Fish Nor Fowl—Dementia With Lewy Bodies Often Missed

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Like DLB, Like AD—Do Oligomers Stir Up the Trouble?

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